Welcome Sarah Stanhope for rejoining your own Biology department.
Sarah Stanhope is a 2020 Magna *** laude graduate from the Biology Department at
Salem State.
While at Salem State, she completed a senior honors thesis
working with Jason Brown, was awarded a Kathy Murphy Endowed
Biology Research Scholarship, and served as the President of the Bio
Society.
Sarah is currently APHD candidate in the Department of Biochemistry at Purdue
University, working under the direction of Vicki Week.
She has presented multiple oral presentations and posters on her work and
has published 4 journal articles including her first authored first first
authored paper in 2023.
Sarah has won numerous awards for her work including an NIHT 32 Drug Discovery
and Development training grant, the Bird Stare Award,
a first place poster presentation award at Hitchhiker's Guide to the Bio
Molecular Galaxy in 2023.
Most excitingly, Sarah just found out this week that she
was awarded A prestigious Kurt Stein NRSAF 31 Individual Predoctoral
Fellowship.
We are so happy to welcome Sarah Stone Stanhope back for her talk entitled
Prolonged Blue light exposure Alters Phototransduction Efficiency and one
Carbon Metabolism Processes in the Drosophila Eye.
Welcome, Sarah.
Thank you so much for that lovely introduction.
I'm so excited to be here with you all today and to share a little bit about my
work of what I've been doing at Purdue.
So I apologize about that.
I'm just going to get the laser pointer out so you guys can see where I'm
directing the conversation.
So today I'm going to be talking to you a little bit about my thesis reach research
entitled Prolonged blue light exposure alters phototransduction efficiency in
one carbon metabolism processes in Drosophila eye.
So just to give you a brief outline today of what I'm going to be going through is
I'd like to start talking a little bit about eye disease and using Drosophila as
a model Organism in case any of you are unfamiliar with that.
I'm then going to touch on some exciting data that we've recently had come out of
the lab focusing on oxidation of phototransduction proteins and machinery.
And then I'm going to finish off with sharing a little bit about my thesis
focus, which is characterizing an enzyme known
as S adenosyl homocystinase or HCI for short, as a redox regulated enzyme.
So to start, I'd like to introduce my lab and one of
the major focuses of our research.
So I'm doing my PhD at Purdue University in Doctor Vicki Week's lab.
And one of our focuses is studying aging and specifically age within the context
of ocular disease, primarily retinal degeneration.
And this is because age is a major risk factor for lots of types of visual
impairment.
So if you look at this graph that I have displayed on the left along the bottom
you'll see years and then on the X axis you'll see percentage of vision loss and
blindness.
And what we know as we age is that once people start to hit around age 60,
we see a drastic increase in the percent of vision loss and blindness.
And as well, studies done by the National Institute of
Health have shown that aging associated ocular disease,
including age-related macular degeneration, cataracts,
diabetic retinopathy and glaucoma are all predicted to double in occurrence by 2050.
And now a little bit about Drosophila and why we use the fruit fly as an aging
model Organism.
So one of the biggest advantages of using Drosophila is that they can age quite
rapidly as opposed to say, doing mouse work.
Whereas if you're studying mice, aging studies may take up to years.
While working with Drosophila, we're comparing this several weeks worth
of aging.
So just to Orient you all a little bit for Drosophila aging,
we count day 10 as relatively young, whereas day 30 to 40 are considered to be
middle-aged and day 50 to 60 are considered to be late aged Drosophila.
And some of the things that we study include visual function.
And what we know with that is that in young flies visual function tends to be
quite good.
However, as the fly ages, we see a drastic decline in their visual
function and that can mean response to light or the health of their eyes.
What we also know is that with age we see increased changes in transcriptional
changes or transcriptomics and we also see increased prevalence of retinal
degeneration.
However, today I'm going to be focusing on a
secondary model apart from aging.
And this is because although aging is a major risk factor,
it's not the only risk factor for the development of eye diseases.
And we have what's called environmental risk factors.
These are things outside of your body that are affecting you and your eye
health.
Some of these can include UV radiations or just natural light exposure, smoking,
bacteria, pollutants, chemicals, or even changes in the temperature.
One thing that environmental risk factors and aging have in common is the
accumulation of reactive oxygen species, abbreviated here as Ros.
Some of the most common active oxygen species that you might be familiar with
could be hydroxyl radicals, abbreviated as OH,
or hydrogen peroxide H2O2.
Now in your cells, these are naturally present and they're
oftentimes used as signaling molecules.
But what can happen is that these can increase in abundance within your cell
due to exogenous or endogenous sources.
Exogenous sources are things outside of you that are affecting you.
Light temperature changes is an oxygen availability,
whereas endogenous sources are things that are coming from within your cells.
This can be like aging, inflammation or infection.
But our cells are quite smart and they developed systems in place that keep
reactive oxygen species in check or in balance.
These are known as antioxidants.
Antioxidants mainly have two main systems, either non enzymatic or enzymatic,
but for the purpose of this talk I would like to focus on our non enzymatic
systems, particularly an antioxidant known as
glutathione.
Glutathione is one of the major antioxidants in your cell and it works to
combat reactive oxygen species.
And you can think of antioxidants and reactive oxygen species as being on a
scale where in general if you're not in a stressful condition these are balanced.
However, if you're under sources of stress,
either exogenous or endogenous, you can get accumulation or buildup of
reactive oxygen species to the point where your antioxidants are no longer
able to balance that.
And when that happens, this is known as oxidative stress.
And oxidative stress can cause damage to macromolecules.
Now, just to focus back on Drosophila and to
give you an overview of the type of tissue that we're working with in my lab,
because we study ocular disease, we're very focused on stress within the
Drosophila eye.
So here on the left, I'm showing you a picture of a male fruit
fly with a focus on its eye.
Now obviously Drosophila in humans have different types of eyes where insects
have a compound eye, and this is what a zoomed in figure of
the Drosophila eye would look like.
And as you can see, these white dots all look like individual
subunits.
Each one of these dots is known as an Amitidium.
An Amitidium is a single repeat is a single unit that houses what we call
Rabdomirs.
If you're unfamiliar with Rabdomirs, you can think of them a photoreceptor
cell for humans.
And on the outside where you see R1 through R6,
these are known as the outer Rabd mirrors, and they're needed for motion vision,
whereas in the center R7, this is needed for color vision.
So each one of these Amitidium have 7 Rabd mirrors, but there's 700,
about 750 of these repeating units that make up the compound eye.
And for the first portion of my talk, I'm going to be introducing some
interesting phenomenon that we've seen within phototransduction machinery.
So I'd like to introduce a simplified version of what the Drosophila
phototransduction cascade looks like.
Now Drosophila phototransduction has been very well characterized through the years
because they are they have incredibly sensitive phototransduction machinery and
Drosophila can actually sense single photons,
which is much faster than our vertebrate rods in our eyes.
But how phototransduction works within Drosophila is that it is initiated by
light and specifically a blue light wavelength.
So the blue light wavelength will activate a light sensing protein,
abbreviated here as RH one.
If any of you have taken upper level biology classes,
you may have heard of a type of protein called AG Protein Coupled Receptor or a
GPCR.
This is characterized by having a transmembrane as well as the three
subunits on the bottom, alpha, beta and gamma.
So what happens when light activates RH One,
its alpha subunit will disassociate and bind to a PLC beta.
This stands for phospholipase C beta, and in Drosophila we call it Norp A.
Now Norp A breaks down lipid molecules like PIP 2 into a secondary messenger
known as DAG.
Another important protein within the phototransduction cascade is known as Ena
D Ena D is a scaffolding protein.
Scaffolding proteins are important because they hold a lot of proteins in
close proximity to each other, so they're able to work together.
So Ena D can bind to NORP A as well as this channel right here known as TRIP.
The trip channel is a calcium ion channel, so when open it allows for calcium to
influx into the cell.
So this would be phototransduction initiation on panel B,
I'm showing an example of phototransduction termination.
So what we would see here is that alpha subunit that was originally from this RH
one light sensing protein disassociates from north A.
And then we have a new player come in.
This is a protein kinase C or PKC for short, known as Ena C in Drosophila.
Ena C needs both DAG, which can be produced by Norp A,
and calcium, which is coming from the influx through
the Chip channel to phosphorylate other protein targets such as Ena D and the
trip channel itself.
Now there's a lot of open-ended questions when it comes to Drosophila termination
as this hasn't been fully characterized yet.
But we do know that Ena C is an important part of the shutting down of the
phototransduction pathway.
So the model that I've been working with in my lab is a prolonged blue light model.
And that's because we know that that blue light wavelength is what activates RH one
into its active form, which can either lead to light response
or endocytosing out of the cell.
And we also know that if the orange wavelength is shown,
that the active form of RH one will then go back to being inactive.
So we know that using prolonged blue light as a model can induce an activation
of the phototransduction cascade.
And this is also important for a lot of our future studies going forward.
But how our blue light machine works in our lab is we would take a vial that has
a number of flies in it and you can put it into these light chambers that we have.
They we can either express, expose the flies to red light or blue
light.
And then we're able to image the fly eye to see if there's any loss of those
Omotidium or Rabdimirs, which would indicate retinal degeneration.
So I'm going to be showing you all a few images today of flies exposed to
different types of light.
But just to remind you of that eye structure,
we have the outer photoreceptor, the outer raptomirs, and the inner,
and you're able to visualize 7 dots through imaging and that is the Amitidium.
As a control for these experiments, we typically use flies that have never
been exposed to light.
This is known as a dark control.
And what you're able to see here is that these armitidium look very healthy with
all 7 rabdomirs present.
But what if we show them a different type of light other than white?
What about red light?
When we show for 8 hours of red light, we also see a very healthy looking eye
structure with no retinal degeneration.
But what about our blue light?
When we show these flies to 8 hours of blue light,
we see mass retinal degeneration with Sometimes when looking for that amitidium,
you're only able to visualize 1 or maybe 2 Rabdimirs.
So we know that in young flies, prolonged blue light will result in
premature retinal degeneration.
Now when I came to summarize some other things we know about a blue light model
from several different papers that have been published by my lab is that blue
light would lead to premature retinal degeneration.
We also know that it leads to increased hydrogen peroxide,
which is one of those common reactive oxygen species.
And we also know that it leads to lipid peroxidation.
And if you're unfamiliar with that term, if you remember back to some of the
courses you may have taken about a phospholipid bilayer,
if you have increased reactive oxygen species,
it can actually break down that lipid bilayer, damaging it.
And we also know that blue light leads to increased oxidative stress because we
were able to measure glutathione within the cell,
which is one of our major antioxidants.
And we know that there's decreased glutathione,
which we can tell is indicating oxidative stress.
So when I came into the lab, we were very focused on different types
of oxidative stress, and particularly my protein fell,
My project fell within the topic of protein damage.
And when we say that oxidative stress can cause protein damage, it's not as if,
you know, holes are appearing in these proteins or
they're all being smashed up.
A lot of times we are actually talking about different types of modifications
that we can be made on these proteins, such as oxidative modifications.
Now, these oxidative modifications can happen
on a variety of different amino acid residues,
but one of the ones that we are specifically interested in are cysteine
residues.
Cysteine residues are important because on their side chain they have a thiol
group which is composed of a sulfur and a hydrogen.
These style groups are quite important because reactive oxygen species are able
to either readily oxidize this style group into another type of modification.
Some some of the ones you might you might be most familiar with are intermolecular
disulfides or intramolecular disulfide bonds.
But these modifications can also be readily reversed back to the original
file.
So you can think of a cysteine as like a molecular switch where it's responsible
for sensing the environment and changing to different types of states.
A lot of this is known as redox signaling.
But what can happen is if you have that imbalance of your active oxygen species,
these cysteine modifications can be further oxidized and you can get what's
known as an irreversible modification.
This can either be sulfonic or sulfonic acid.
And these types are what lead to cellular damage because they can no longer be
reverted back to the cysteine's original file.
So one of the major questions that we asked upon joining the lab is what
proteins specifically are susceptible to this type of oxidative damage at their
cysteine residues.
And how we did that is through a technique called redox profiling of the
proteome.
So what we did here is we exposed flies to blue light,
we manually dissected their eyes, extracted the proteins out,
and then performed a little bit of some complicated labeling.
So I'm going to just briefly touch on how this labeling works,
but if you have more specific questions about it later, I'd be happy to take them.
Or if you have one that you'd like to e-mail, that's fine as well.
But how this labeling works is once we extract the proteins,
if a cysteine is reduced, meaning it has that original thiol group,
then A tag can bind to it.
But if a cysteine is in a different type of oxidative modification that meaning
that it's no longer in that original thiol state, A tag cannot bind.
So we do this type of labeling with all of the proteins we extracted from the eye,
and then we're able to identify which proteins we isolated using liquid
chromatography mass spectrometry.
And we do this with an incredible group at the Indiana School of Medicine in
Indianapolis.
And essentially what this data is looking at is changes in cysteine availability
through tag abundance.
So how this is going to look is if you have a decreased amount of these tags,
this is going to correlate with potential oxidation because that means a cysteine
was not reduced or wasn't available to bind that tag.
So you're going to see less of it.
Whereas if you see an increase in the number of tags,
this could correlate with potential reduction because that means the
cysteines were in their original thiol state and the tag was able to bind.
So we're able to summarize all of this proteomic data in a volcano plot.
If you're unfamiliar with looking at volcano plots, to simplify it here,
anything that is shown on the left side is going to be considered significantly
oxidized, whereas anything that's shown on the
right side is going to be considered significantly reduced.
So when we were looking at this data, we were able to identify that blue light
resulted in significant oxidation of several phototransduction machinery,
including some of those important key players that I mentioned earlier like Ena
C and Ena D, which are also shown here in this
repeated schematic that I showed before of Drosophila phototransduction.
So just to show you again Ena C, which is that protein kinase C we
identified as having several oxidative modifications and again Ena CS role is to
use both DAG and calcium to phosphorylate other protein targets.
We also identified oxidation of that important scaffolding protein Ena D,
which is that protein responsible for holding all of the other
phototransduction machinery in close proximity to one another.
Now it's one thing to identify these oxidative modifications,
but we don't really know what that means in a functional sense.
So one of the follow up questions that we had from this study is if cysteine
availability changes in these phototransduction protein,
then what is happening to the efficiency of phototransduction under these
conditions, meaning the blue light exposure,
Is there a functional consequence for this oxidation that we're seeing?
And one way that we're able to do this is or evaluate these functional consequences
is through a technique called Electro retinograms or Ergs.
Now Ergs measure the electrical activity of the retina in response to light.
So this can actually be done in live flies.
So over here on the left, I'm showing a family that has been
mounted to a slide and inserted is a metal electrode into the fly retina.
And that's how you're able to measure those electrical responses.
So before I go into what this data looked like,
I just want to prepare you for what it will look like.
So when we're looking at ER GS, there are three main steps that we're
looking for.
We're looking for this on transient, which is called the hyperpolarization of
the neuron.
We're also looking at receptor potential.
So this is depolarization.
And then we're looking for the sharp peak at the end,
which is known as the off term, the off transient.
This is the termination response.
You will then see this return back to baseline and it will repeat again when
showing light.
So one of the questions that we had is for flies that were exposed to blue light,
how does this erg change with increased light intensity?
So this data can be a lot to look at upfront.
So I'm going to walk you through it quite slowly,
and I'd like to start by noting that this is in a negative log scale.
What this means for the data is that as you read left to right,
the light intensity increases, which means that these flies are being
shown brighter and brighter and brighter light.
And as I mentioned before, Drosophila phototransduction is a much
faster process than humans are, and they can sense even a single photon.
So all though we're seeing an increase in the light intensity,
if I were to show this to you, it would almost be as if there was no
change in the light because we can't respond as fast as the fruit flies can.
So we're going to start here along the bottom with this white light illumination.
So each of this, each of these ER GS is an individual fly.
This is done on live flies and that were either exposed to white light or to blue
light, which is our experimental prior to the
ERG.
Flies are dark adapted so they're kept in a space with no light.
That way when you're doing this assay, they are reporting from a baseline and
it's not like they're already responding to light prior to the beginning of the
erg.
But what we're looking for in this data is those 3 distinct portions again the on
transient, the receptor potential in the termination
and for flies that are our control, which means just the white light
illumination.
We start to see that response around negative log 5 where you can see the peak
at the top, this little drop off, which is the receptor potential that off
transient and then return to baseline.
And as you if you notice as the light intensity increases,
meaning as we go more towards the right, this drop down which we know is amplitude
keeps going further down, meaning that the amplitude is
proportional to the light intensity.
So we expect this to continuously the we expect the amplitude to go down further
as we read the graph.
Now what about blue light?
So this is testing that functional consequence.
Does blue light change how the flies respond to light?
So if we look at negative log flat 5 where we saw the white light illumination
response starting, we don't see anything for flies that were
exposed to blue light.
And in fact we don't start to see a full response until we get to about negative
log 3 1/2.
So their response is starting much later.
And another important fact that we see when looking at this data is that their
amplitude, this down portion is not proportional to
the light free intensity as it was in our white light control.
So this graph down the bottom where we're showing the same negative log scale as we
are up here and we're showing that amplitude,
which is this drop down on our X axis.
What we can see in the summarized data is that we have a shift in response time,
so delayed response.
And also if you notice that this blue line runs below the white,
that is showing that decreased amplitude.
So to summarize this data or like what does this data mean?
The ERG is showing decreased sensitivity and dampened amplitude.
This means that blue light in fact dampens the flies electrical response to
light.
And what we're seeing is something that's very similar to light adaptation.
This means that although the flies were kept in the dark prior to the erg,
they respond to the first point of light as if they've been responding to light
constantly without shopping.
The next question might be, why is this important?
So having the ability to shut the pathway down or turn it off,
it's just as important as being able to turn one on or initiate one to begin
without terminating the signaling event.
The eye is not able to adapt to different intensities of light,
and this means that your phototransduction pathway can become
saturated.
So although there's nothing physically wrong with the eye,
it is functionally blind.
So when we were thinking about this data and knowing that we saw oxidation of
several different phototransduction proteins,
we wanted to know which of these proteins might contribute to this ERG phenotype
that we're seeing, which protein might be responsible.
And when reading about these proteins, we started to really focus on Ena C or
that protein kinase C.
So again, that's responsible for phosphorylating
one of the substrates is a known substrate for Ena D and potentially the
trip channel.
But there are also substrates that we don't know.
And here on the right, I'm showing a predicted Drosophila alpha
fold structure of Ena C And through our proteomic profiling,
we found that three individual cysteines had oxidative modifications.
And what's interesting is that each of these oxidative modifications are
clustered together within a single domain of Ena C and this happens to be in the
DAG binding domain.
So Ena C needs to bind DAG and calcium in order to perform its phosphorylation
activity.
So we had a question of if the cysteines are oxidized,
how might that change ENAC activity in the eye.
And so we started digging in the literature and we mammalian PKC's.
So human PKC's and Drosophila PKC's are highly conserved and they have the same
domain regions such as a regulatory domain,
which includes that DAG binding domain as well as its kinase domain,
its catalytic domain.
And what we were able to find from previous studies that came out in the 90s
was that oxidation of cysteines in that DAG binding domain actually led to
activation of the PKC regardless of whether or not DAG was bound.
So our current working hypothesis for this the project is that we think that
redox signaling or oxidative stress activates ENAC,
that protein kinase C independent of the PLC beta mediated DAG production or the
calcium influx from the trip channel.
And this might be what's enabling sustained light up adaptation following
those bright light intensities.
And some new data that we've just received as well is showing that if you
knockout or remove Ena C from the fly eye, they actually have increased intensity
response after blue light exposure.
So we are able to rescue that delayed response time.
Now some of the remaining questions that we have for this project is how does Ena
C contribute to these two intense light response?
So we're thinking how could Ena C maybe change its activity in order to alter the
termination of our phototransduction cascade?
And also how does blue light affect Ena C?
How might this change its structure?
We saw that those cysteine modifications are made within a single domain that DAG
domain.
Some of the questions that we have are might these modifications change how that
domain looks?
Could that domain unfold giving in ACA different act or a change in activity?
Another question is does blue light make in AC hyperactive or could blue light
impair protein protein interactions?
As we know Ena D that important scaffolding protein holds all of the
phototransduction machinery in close proximity.
So if we have oxidation of that protein kinase C,
could that prevent it from interacting with the scaffolding protein?
Another big question are what are Ena CS phosphorylation substrates?
Only some are known, but not all.
And one other big question that we have is what could initiate this type of redox
signaling.
Reactive oxygen species aren't just floating around in the cell and they have
to be produced locally, which means a protein has to be in close
proximity to Ena C, producing reactive oxygen species in
order for Ena C to become oxidized in the area that we're seeing now.
This is currently an open thesis project for a PhD student at my lab at Purdue
University.
So if you're interested in grad school or this project sounds interesting to you,
I'd be happy to take questions at the end or you can e-mail me for further
information.
But for the last portion of my talk, I would like to touch on what I have been
studying for my thesis work, which is characterizing S adenosyl
homocystinase or AHCY as a redox regulated enzyme.
So although blue light heavily affected phototransduction machinery,
it was the what was interesting is that actually our most significantly oxidized
protein that was identified in Drosophila eyes and cultured cell lines was a
metabolic enzyme known as a HCYAHCY is an important enzyme when we're thinking
about epigenetic or epigenetic modifications.
And for those of you that are unfamiliar with epigenetics or the modifications
applications that can take place, I'm just going to show you a quick
schematic of what this might look like.
So I'm sure many people know what chromosomes are, but when you unwind them,
we have another term for it called chromatin.
And chromatin is made-up of tightly wound DNA around histone proteins.
So down here the purple is the tightly wrapped DNA where the blue is the
histones.
Now histone proteins are quite important.
They have this feature called a histone tail.
Now dependent on what type of modification is taking place,
this can have different ramifications for DNA and histones.
One of the most prevalent modifications that I'm going to be talking about for
the majority of this talk is called methylation.
And for methylation to occur, you need the addition of a methyl group.
So what's very interesting about histones is if they are modified,
say by a methyl group at a specific spot in their histone tails,
it can cause them the they can cause them to separate and for the DNA to become
accessible.
Sometimes this will be taught to people as beads on a string where it can either
be tightly wound or they can separate where the histones are then spaced out.
Your DNA is accessible in between and this can lead for active transcription of
the genes and that's located there.
But what's important is that if the modification is made on a different
location of the histone tail, they can actually revert back to being
inaccessible or tightly wound DNA where your gene expression is then inactive.
And you might be thinking, how does this relate to that protein?
I mentioned Hcy, and that's because Hcy functions in a
very important branch of metabolism known as one carbon metabolism.
One carbon metabolism is what produces this universal methyl donor known as Sam.
So how this process works is that you have your essential amino acid,
methionine gets broken down to generate the metabolite Sam.
This M on the end is for the meth, the methyl group.
So this is a universal methyl donor for all methylation reactions in the cell,
including DNA methylation, mRNA methylation and histone methylation.
And it's highly abundant within your cells and it's the second highest used
cofactor after ATP.
And what's important about Sam is once that methyl group gets used by proteins
known as methyl transferases, it generates A byproduct metabolite known
as S Adenosyl homocysteine or SAHSAH, has an opposing role to Sam as it's
actually an inhibitor of methyl transferases.
So methyl transferases can either bind Sam to use its methyl group to methylate
another substrate or methyl transferases can bind SAH.
And if they find SAH, then they are no longer able to methylate
other proteins.
And when we talk about Sam and SCH, we normally refer to them as a ratio,
and this can also be termed the methylation index.
And if this ratio is disrupted, it can actually prevent all methylation
reactions within your cell.
And HCY has a very important role in this process by its interaction with SAH.
Now HCY is one of the most conserved enzymes across all living species.
And here I'm showing just a rough domain map of HCY where you have it substrate
binding domain, it's hinge region which allows it to move
it's and it's NAD binding domain.
So HCY functions as a **** tetramer.
So here in panel D I'm showing a predicted structure of what a monomer
would look like.
So one of its subunits and four of these monomer subunits will come together to
form the **** tetramer, which is then it's able to be in its
active form and facilitate local transmethylation reactions.
And one of the most important things about HCY is that across all living
species, it is the only enzyme to hydrolyze or
breakdown that inhibitor metabolite SAH.
So this is just a structure of SAH.
And when HCY breaks it down, it breaks it down into two components
known as homocysteine or adenosine.
Adenosine is very important for nucleic acids,
but homocysteine is actually of hot commodity within your cell and it can get
used in a variety of ways.
Two of the most common ways that homocysteine is needed in the cell is
either through the transulfuration pathway,
which this pathway uses homocysteine to convert it into cysteine,
that amino acid residue, and this can then be used for either
protein synthesis or antioxidant production.
Like glutathione, which I mentioned earlier,
Homocysteine can also be used in the remethylation cycle.
This means that homocysteine would get further broken down and it's used to
regenerate methionine to restart that one carbon metabolism pathway.
Now, although I'm very interested in HCY in
the context of ocular disease, there are also a variety of diseases that
are associated with HCY, including several different types of
cardio, cardiovascular diseases, metabolic disorders.
HCY has also been studied as an antiviral target for virus infections as well as a
tumor suppressor.
Now for us, when we're thinking about a HCY,
we know that it was the most significantly oxidized protein in our
proteomic data set.
And we also identified a single oxidative modification at a residue known as
cysteine 195, which I'll be referring to as C195 for
the rest of the talk.
Cysteine 195 is in a very important location for AHCY and it's located near
its catalytic center.
And a study that was done in the 90s, which is an in vitro study,
meaning that this protein wasn't taken from a whole Organism like Drosophila.
It was produced recombinantly using bacteria.
They found that if you modified that recombinant protein at location C195 to a
different amino acid such as a serine, that this revealed a significant decrease
in catalytic turnover.
And catalytic turnover is a fancy word for saying that if you mutate the
cysteine, HCY as a protein has a very difficult
time releasing its substrate SAH, meaning that it's catalytic efficiency is
much lower because instead of being able to bind SAH,
break it down and release the products.
If you mutate this residue, SCH almost gets stuck and it's not able
to be broken down.
So what we wanted to know in vivo for the Drosophila eye is does this modification
have that functional consequence?
What might this oxidative modification be doing to the protein activity?
Although we don't have an activity assay yet for Hcy, we have the next best thing,
which is being able to measure its substrate abundance within the eye.
So just to remind you again of that pathway,
we have methionine being used to generate Sam, that universal methyl donor.
So it will get taken up by a methyl transferase,
which then is used to methylate a substrate.
When this occurs, it generates SAH which can inhibit methyl
transferases, and SAH can be broken down by a HCY.
So what I'm showing you here is targeted metabolite analysis.
This means that we had two subsets of flies,
either flies that were exposed to normal white light or flies that were exposed to
8 hours of blue light, which we then dissected their heads,
extracted the metabolites and quantified how much of each metabolite was present.
So when we look at Sam, there is no significant change between
flies that were showing or shown to 8 hours of blue light.
Their Sam abundance, which again is here in the pathway,
is not changed.
However, what we see with eight hours of blue
light is a significant increase in the metabolite SAH,
which indicates that Hcy may not be functioning properly upon blue light
exposure.
And as I mentioned earlier, normally we talk about Sam and SAH as a
ratio to one another.
And what we can tell through this also is that blue light significantly reduces the
Sam to SAH ratio.
And this disruption of the methylation index can prevent the methylation
reactions in the cell, because too much accumulation of SAH can
lead to the inhibition of methyl transferases,
which could subsequently alter your epigenetic expression.
Or changes within your gene expression.
So our current working model for this is that a HCY is a redox regulated enzyme,
meaning that under normal physiological conditions Sam is used by methyl
transferases to produce SAH which can then be broken down into two separate
products such as adenosine and homocysteine by a HCY.
However, under stress conditions like blue light,
these reactive oxygen species make that oxidative modification at C195 leading to
possibly inactivation of AHCY mean, which means you don't get the production
of adenosine and homocysteine, but instead increased abundance of SAH,
which we then think is inhibiting methyl transferases.
Now one thing to keep in mind when we do redox proteomic profiling,
we are identifying 10s of thousands of proteins at a time.
And we, when you're analyzing these data sets
using bioinformatics, we try our best to identify true or
significant hits while eliminating anything that might be considered an
artifact or a false positive.
So it's very important that when you do an omics data set that you have a second
way of validating your findings.
And one way that we've done this in our lab is through a different type of
labeling that can then be visualized by Western blot.
So here I'm going to walk you through how this type of labeling works.
And here's a schematic of HCY with its cysteine residue at position C195.
And this is in a reduced state.
If your cysteine is in a reduced state, it can bind to A tag.
This tag is known as MMM PEG 24.
This is a compound that can increase proteins molecular weight by 1.
24 kilodolins per cysteine residue.
But if you have a cysteine that's in an oxidative modification like I showed
earlier, your tag won't bind, meaning that your molecular weight of
your protein will remain unchanged.
Also, if you mutate that cysteine residue to
say a serine, where the serine no longer has that thiol
side chain and said it has an 0, the tag also won't be able to bind.
So you will not have a change in the protein molecular weight.
So what does this look like on a block?
So I'm going to walk you through slowly through each lane.
Here we're focusing on sizes between 75K daltons and 63K daltons.
I'm showing a wild type HCY, an HCY with at that cysteine 195.
We've mutated it to an alanine or we've mutated it to a serine.
So this first 3 where it says HCY ox C 195 ox or C195 S ox.
This is proteins that have been extracted from Drosophila cells that have been
exposed to oxidizing reagents, which means that we are forcing available
cysteines to take on an oxidative modification.
Or this next three, we're looking at the wild type or either
of the mutants that have been reduced, meaning they have been exposed to
reducing agent that forces those cysteines to be in their original thiol
state.
And one thing that we noticed so down these last three are true experimental
samples where we're looking for that weight increase with the MMM PEG tag
being bound.
And what we can see here is that this wild type AHCY meaning that it has
cysteine 195 when incubated with that MMM PEG tag runs at a higher molecular weight
just slightly by what looks to be around 1 kilodol and higher than if you mutate
the cysteine 195 to either an alanine or a serine,
indicating that a HCY at position C 195 is the major oxidized cysteine residue
for this protein.
Now we still, I still have a lot of work to do on this
project and we have several open questions that are currently being worked
on.
And the first part of the question would be how would an oxidative modification or
a mutation at cysteine 195 affect HCI activity.
So as I mentioned earlier, we didn't have an activity assay at the
time, which is why we measured its substrate
abundance.
So I'm working to characterize HCI activity using deabsorption electrospray
ionization mass spectrometry.
This is abbreviated as DESI MISS.
This is a newer form of enzyme activity assay,
which is known as a high throughput and highly sensitive way of measuring enzyme
activity.
Our second question is what genes are directly regulated by HCY in the eye?
This is currently in progress and we're doing a large scale sequencing experiment
using cut and run.
If you're not familiar with cut and run, this is a way to pull down DNA that is in
either in close proximity to or bound by HCY.
A third question is if HTI is depleted, meaning that you either knock it down or
knock it out in the eye.
We think that this would correlate with increased SAH abundance,
but we want to know how would that change gene expression in the eye 'cause
remember, if you have an increase in SAH, you can potentially be inhibiting your
methyl transferases.
So how we're answering this question is doing another type of large sequencing
experiment known as RNA seq.
And this is when you sequence all of the RNA with and in the photoreceptor cells
that do not have a HCY.
And these are things that I'm currently doing in my lab.
So before I end my talk today, I would like to just show a little bit
about the Department of Biochemistry at Purdue in case there are any students in
their upper level of their undergraduate that are interested in Graduate School.
So our biochemistry department has many different focuses of research,
including metabolic and natural product biochemistry, omics,
so that some of the things that I've shown you today like genomics and
proteomic data, as well as cancer biochemistry,
epigenetics and gene expression, as well as structural biology.
And if you're interested in applying to Graduate School, Purdue does offer a PhD.
We have 35 week rotations.
The application deadline would be November 15th.
We do not require a GRE and PhD programs do offer a stipend,
which is currently at 33,000.
And if you are interested, I've also provided a link to our
department.
And with that, I would like to thank the organizers of
the Darwin Festival for inviting me.
I would like to also thank my thesis committee,
members of the current graduate students that I work with,
which are shown here in this photo, as well as graduate students that have
graduated, and of course, the funding that is provided through my
lab.
And with that, I'd be happy to take any questions.
Thank you Sarah, great talk.
Nice to see you again.
You as well.
We come from a line that is the mammals where regeneration and nerve tissue is
very seldom seen.
Yes, in in Drosophila can retinal cells
regenerate?
So they can't regenerate.
So these are still post mitotic cells.
But what we have seen through transcriptomic data that we've done in
our lab is that when the cells are, when the photoreceptor cells,
which are post mitotic cells are under stress with either with aging or blue
light before they die, they do re enter the cell cycle.
And it's almost as if those neurons are trying to save themselves,
but they never quite get there.
Great talk.
Sarah, there's actually a question from one of
my students about your final slide and I cheated, Sarah.
I took a screenshot.
So I'll e-mail that to you, the student who just asked that question
and a question for our undergraduate.
Sarah, when you were here at Salem State, how did you look into which graduate
programs might be be good for you?
And how did you decide on Purdue?
Yeah, so when I was at Salem State, I did multiple semesters of research with
Doctor Jason Brown, which I know unfortunately couldn't be
here today.
But he was a great help when it came to looking at what was available to me for
Graduate School and different types of programs and things like that.
But when I was looking for graduate programs,
there were things that were important to me like the ability to do rotations,
which means that you basically get a trial run for whatever or like a several
week.
In a different labs and you can see what is the right fit for you.
So I knew I wanted to be in a program that offered that and I also wanted to be
in an area that had a lower cost of living.
That way you can make your grad stipend work no matter what.
And one other important thing is when you're looking at programs,
make sure that you are interested in more than one faculties research.
It's important that you have a variety of options.
That way, if you get into a lab and on paper,
it seemed perfect for you, but maybe it wasn't the right fit once
you got there, you weren't stuck in a program where you
didn't think anything else was as interesting.
Thank you.
I have a question for you.
The next question is Sarah, for us, where is blue light exposure most common?
So for humans, when are we going to encounter blue light?
Any time that you are under LED light, there are blue light wavelengths within
that.
Of course, our blue light model is an extreme model.
That way we can see the direct effects of blue light.
But anytime you're looking at your computer screen, your phone,
or even the lights in your classrooms, those all have blue light wavelengths in
them, right?
Thanks, Paul.
Are you going to do the next one?
I don't have an X1, do you?
Oh, I All right, so I have an extra somehow.
So a question from one of our upper level students.
Sarah, thanking you for a very interesting and
informative talk.
Some general questions and comments from you about being a PhD student.
What's it like?
And to answer the students interest because she's interested in a PhD program.
So yeah, what's it like?
Well, it's very, it's busy, but every day is so interesting because I
get to learn something new constantly, whether that's new techniques or new
areas of research.
Sometimes the best part of my day is if when new data comes in and you are at
that moment, that first person or the only person to
know this type of information before you're able to put it out there,
and then sometimes the best feeling in the world.
It's pretty different, I would imagine.
Yes, Sarah, are humans damaged by blue light and
should we try to avoid it or filter it somehow?
So there it is known that prolonged exposure to a lot of blue light is
damaging to your eye.
I don't know if there's any way to really avoid it.
They do sell those blue light filtered glasses that you can buy,
but those haven't really been shown to have any type of effect.
But I think just monitoring how much screen time you're doing throughout your
day is important.
Thanks, Sarah.
Your last slide, you showed us how a student might find
out more information about a PhD program and I'll, I'll make that available.
As I, as I said, I took a screenshot of that.
But if a student, and there are a few of them,
judging by the questions of thinking about your type of PhD research,
if you cast your mind back to being here as a junior and senior,
what courses would you recommend they take?
I So I guess it would depend on what type of program they're looking into.
If you're leaning more towards a biology PhD or a microbiology PhD,
taking those upper level courses like microbiology would probably be very
important for you.
If you're interested in something more similar to what I'm doing with
biochemistry.
If you're a biology major taking extra chemistry classes,
really paying attention in organic chemistry and taking biological chemistry
is important, but also if you have the ability to get
research experience that also makes a world of difference when applying to a
program.
Great thanks.
Oh there is one more I can see.
Oh should I read it or?
Oh I had to Scroll down.
OK.
Is there a cut off for blue light wavelength beyond which damage does not
occur or is minimal?
So what we've done in the Drosophila eye is we've done various time courses.
So for the studies here, I showed 8 hours and we know that 8 hours
causes mass retinal degeneration.
But if we do something smaller like 3 hours,
which is what we consider to be an acute stress,
there is some damage to the rabdomers, meaning that some of those might be
missing within those images, but not to the same extent as 8 hours.
So time does matter and is it a single wavelength or do you use multiple
wavelength?
This is a single wavelength.
Sarah, a question from I'm sure you took Cell
Biology with Doctor Nelson Scott Gale.
Yes.
Is macular degeneration correlated with increased exposure to blue light exposure?
So I know that age-related macular degeneration is more,
you have more of a genetic predisposition.
I don't know if blue light might correlate with an increased chance of
age-related macular degeneration.
I'm not sure on that one.
Right.
And another student question, how do you think your research with
Drosophila will impact your future research endeavors?
Well, I think that working with Drosophila
means that you have a lot of genetic background.
So Drosophila is a incredible model Organism for a lot of different types of
genetic based techniques, but how it will influence or impact my
future career goals?
I think one of the nice things about doing a PhD is you're not going to get or
lose a job based on what model Organism you did your PhD.
And I don't think it's a limiting factor.
I think it's based on what you're interested in at the time.
Thank you Sarah.
We'll make this the final question given everybody in the audience that we have
another tool in just over 20 minutes by whether or not a question Sarah that you
probably had asked of you in one of the seminars you forces here at Salem State.
Where do you see yourself in 10 years time?
In 10 years time?
I so my career goal after finishing my PhD is industry work.
So I hope that I'll be working somewhere at maybe a proteomic base or protein
based drug discovery company, Right.
Well, thank you very, very much for joining us.
And you told me earlier before we came online with everyone hoping to graduate
in how soon spring 2025 S next spring.
Thanks very much Sarah, take care and everybody,
thank you for having me.
Thank you, Sarah.
Bye.