My name is Russell DeBose-Boyd,
and I'm from the Department of Molecular Genetics
at the University of Texas Southwestern Medical Center in Dallas, Texas.
In Part 2 of my talk,
I'll talk about Schnyder corneal dystrophy, or SCD,
and the importance of the protein called UBIAD
in the regulation of cholesterol.
So, what is Schnyder corneal dystrophy?
SCD is a rare autosomal dominant eye disease
that is characterized by the bilateral of opacification of the cornea.
This corneal opacification associated with SCD
is progressive,
it can be detected in patients as young as 10 years of age,
and it reduces visual acuity.
That typically requires corneal transplantation
in more than 50% of patients that are over 50 years of age.
Now, the biochemical analysis of SCD
revealed that cholesterol markedly accumulates
in the corneas of SCD patients,
which indicates that dysregulation of corneal...
of cholesterol metabolism
contributes to the pathology of this disease.
Now, mutations in the gene encoding UBIAD
cause SCD,
and this was indicated by these two early papers in 2007.
It turns out that 25 missense mutations
that alter 21 amino acids in UBIAD
have been identified in about 50 SCD families.
So, what is the function of UBIAD?
So, UBIAD actually stands for UbiA prenyltransferase domain-containing protein-1.
So, UBIAD is a membrane protein, as illustrated here,
with 8-10 transmembrane domains.
And it belongs to this huge family of prenyltransferases
called UbiA prenyltransferases.
These UbiA prenyltransferases are found in all forms of life,
from bacteria to man,
and they all transfer prenyl groups to aromatic acceptors,
generating a wide range of molecules such as
ubiquinones, chlorophylls, heme,
vitamin E, and vitamin K.
So, what does UBIAD do?
So, shown here is another depiction
of the cholesterol biosynthetic pathway
that I talked about in Part 1 of my talk.
So, UBIAD actually utilizes
one of the products of the cholesterol synthetic pathway
called geranylgeranyl pyrophosphate,
or GGPP for short.
And what UBIAD does is it transfers
the geranylgeranyl moiety from GGPP to menadione,
or a form of vitamin K called vitamin K3,
to produce menaquinone-4, or vitamin K2.
So, UBIAD uses GGPP as a substrate
to synthesize a form of vitamin K2
called menaquinone-4 or MK-4.
Now, shown here is a crystal structure
of a bacterial UbiA prenyltransferase.
And on this crystal structure,
we've been able to map the amino acids that are...
that correspond to SCD-associated mutations
in human UBIAD1.
And what these studies reveal is that
all of these SCD-associated mutations in UBIAD1
cluster around the active site
of the UbiA prenyltransferases,
which indicates that perhaps these SCD-associated mutations
somehow disrupt the recognition of the enzyme
for the isoprenyl substrate.
Now, our interest in UBIAD1
and its role as a sensor of GGPP
stems from the fact that early studies in our laboratory
had indicated that GGPP
mediates or augments the accelerate...
sterol-accelerated ERAD of HMG CoA reductase
that we covered in the first part of my talk.
So, as shown here, HMG CoA reductase
somehow senses sterols, either through direct or indirect mechanisms.
This sensing causes the reductase
to bind to Insig proteins
that are, again, associated with E3 and E2 ubiquitin ligases.
This bridge... this actually bridges reductase to the ubiquitin complex,
resulting in ubiquitination of reductase membrane domain.
And this ubiquitination causes the reductase to be now recognized
by proteasomes and degraded.
And it turns out in early studies
we discovered that this degradation of ubiquitinated reductase
is augmented by GGPP.
However, the mechanism for this GGPP-enhanced ERAD
of the reductase was completely unknown.
Okay, the observation that GGPP
augments the ERAD of HMG CoA reductase,
together with the observations that mutations in UBIAD
which cause SCD
are predicted to disrupt its binding of GGPP...
this prompted us to appraise a role for UBIAD in the ERAD of reductase.
And that role of UBIAD in reductase ERAD
is summarized in this slide.
So, as I mentioned earlier, the accumulation of sterols
causes reductase to become...
to bind Insigs and then become ubiquitinated.
We believe once the reductase is ubiquitinated,
a subset of reductase molecules bind to UBIAD1.
Importantly, the binding of reductase to UBIAD
blocks reductase degradation,
and it protects it from degradation.
Until sufficient levels of GGPP are produced,
which in turn bind to UBIAD,
and the binding of GGPP to UBIAD
results in the dissociation of this reductase/UBIAD complex,
and that dissociation then allows a subsequent
membrane extraction, dislocation, and proteasomal degradation
of the reductase.
In fact, it appears as if UBIAD is an inhibitor
of reductase ERAD, and this ERAD is...
this inhibition is relieved by GGPP binding.
And our first realization came when we decided
to study the subcellular localization of UBIAD in cells,
and that localization is depicted here.
So, here we've localized UBIAD in cells at...
that are cultured under conditions
in which the cells are replete with both cholesterol and GGPP.
And we were surprised to find that UBIAD
localized to the Golgi apparatus.
However, when we treated these cells with a statin
to deplete the intracellular stores of GGPP
and other non-sterol isoprenoids,
we noticed that UBIAD relocalized
from the Golgi apparatus to the endoplasmic reticulum.
We could restore the Golgi localization of UBIAD
by simply adding GGPP to these statin-treated cells.
So, that allowed us to add another level to our model.
And that is what...
what's happening under our conditions is that GGPP binding to UBIAD
not only causes UBIAD to dissociate from the reductase,
but it also allows UBIAD
to transport from the ER to the Golgi.
And then we decided to focus on asparagine 102 of UBIAD.
This is the most frequently mutated residue in SCD patients.
And as shown here... this is a model of the human UBIAD active site
that's built upon the bacterial crystal structure
of the enzyme,
and it shows that this asparagine 102,
shown here,
actually mediates the binding of the isoprenyl substrate.
So, one would predict that the mutation...
and I should point out the mutation that's found in the human UBIAD
is an asparagine 102 to S mutation,
and we predict that this mutation
would disrupt the binding of UBIAD to GGPP.
Indeed, mutation of this asparagine 102 abolishes
-- completely abolishes --
enzymatic activity of the UBIAD protein.
It also turns out that this N102S mutation
blocks the ER-to-Golgi transport of UBIAD,
and that's depicted in this experiment.
So, shown on the left is wild type UBIAD
in cells that are treated with a statin.
And you can see that the protein
is sequestered within the ER.
However, we can induce the translocation
of this UBIAD to the Golgi
by simply adding GGPP to these statin-treated cells.
As you can see, the N102S
-- this SCD-associated N102S version of UBIAD --
is sequestered in the ER,
just like the wild type protein.
However, it's completely refractory to this GGPP-induced
transport to the Golgi,
presumably because it can no longer bind to its substrate,
GGPP,
owing to this mutation that converts asparagine 102 to serine.
Now, in subsequent experiments,
we've discovered that this UBIAD N102S
continues to bind to the reductase in the presence of sterols,
continues to block reductase degradation.
However, because it can no longer
sense the levels of GGPP within cells,
it never dissociates from the reductase.
It remains associated with reductase;
it remains sequestered in the ER.
And this sequestration and
GGPP-resistant binding
leads to a block in the reductase degradation.
We believe that this block in reductase degradation
contributes to the accumulation of cholesterol that is...
characterizes the SCD.
And it turns out that all SCD-associated mutants of UBIAD...
and there are 19 additional mutants to the N102S mutation...
they're all sequestered in the ER,
they're all defective in transport from the ER to the Golgi,
and they all block the ERAD of HMG CoA reductase
in a dominant negative fashion.
So, our next question was,
does this SCD-associated UBIAD
modulate the ERAD of the reductase in whole animals, in mice?
So, for that purpose, we created a knockin mouse
in which we changed,
in this case, the N100 correspond...
in mouse UBIAD corresponding to the human N102.
So, we simply created an SCD mutation in mice
by changing this N100 to a serine residue.
So, this slide kind of depicts our breeding strategy.
So, we actually introduced this N100 mutation into mice,
and we took male and female mice that would have
one good copy of the UBIAD and one of...
one copy that harbors the N100S mutation.
And we bred male and female mice that are heterozygous...
it's called heterozygous...
for this UBIAD knockin.
And this mating led to three genotypes:
a wild type;
a heterozygous, which, again, contains one wild type copy of the UBIAD gene
and one N102S knockin copy;
and then a homozygous, which could...
both copies are the N100S mutation.
So, once we generated these animals,
we then isolated these animals
and took various tissues
and did immunoblot analysis of HMG CoA reductase.
And that's shown in the bottom part of the slide.
And you can see for the liver...
we can see that the reductase levels are low in the wild type animals,
they're increased in the animals that have
one defective copy of the UBIAD gene,
and they accumulate even further
in animals that have two copies
of the UBIAD N100S mutation.
Now, you can see a similar accumulation of reductase
in the eye, testes, spleen, and pancreas.
So, what these results indicate to us is that,
just like in cultured cells...
that this SCD-associated mutation,
this N100S mutation,
in the UBIAD gene leads to a block in reductase degradation,
and this block in reductase degradation leads to a marked accumulation
of the reductase protein.
And it indicates that the ERAD of the reductase
is blocked in these knockin mice.
So, our next question was,
does this block in the reductase ERAD
contribute to the opacification that we see in...
of the cornea that we see in human SCD patients?
So, this experiment shows the analysis
of eyes of either wild type or UBIAD knockin mice
after they've aged about 50 weeks.
And what you can see after 50 weeks of aging...
in both the left and the right eye,
we can see signs of corneal opacification.
And I should point out that the corneas of these animals
not only exhibit this corneal opacification,
but they also markedly accumulate HMG CoA reductase protein,
and they also have other hallmarks of sterol overaccumulation.
They have increased cholesterol,
and they have increased levels of genes
that are required for cholesterol transport.
These are typical hallmarks of sterol accumulation.
So, indeed, these studies
revealed that the mouse N100S,
just like human N102S,
resists this GGPP-induced release from reductase
and blocks its ERAD.
So, under normal conditions is shown here,
this GGPP sensing that's mediated by UBIAD1
is responsible for the maintenance of cholesterol homeostasis.
However, in SCD mice,
this sensing of GGPP is actually disrupted.
And when it's disrupted,
the UBIAD always associates with reductase,
thereby blocking its ER-associated degradation.
And this block in degradation leads to enhanced synthesis
and accumulation of cholesterol,
and this contributes to the corneal opacification
that characterizes SCD,
and perhaps other phenotypes that have yet to be discovered.
So, in summary,
what we've discovered is that this UBIAD protein
continuously cycles between the ER and the Golgi.
Once it senses a decline of GGPP levels in the ER,
it then becomes trapped in the ER,
where it then can bind to the reductase
and block its degradation.
And this block in degradation allows
for continued synthesis of GGPP,
even though the cells have plenty of sterol.
So, what really controls this is the level of GGPP
within the ER membrane.
So, once levels of GGPP
accumulate to sufficient levels,
it binds to UBIAD,
and that allows UBIAD to dissociate from reductase,
and it translocates to the Golgi.
We believe that this UBIAD-mediated sensing of GGPP
becomes disrupted in SCD.
And this disruption results in the inhibition
of the reductase ERAD.
And this inhibition of reductase ERAD
contributes to the sterol overaccumulation
that characterizes SCD.
So, finally, I'd like to acknowledge those that have...
that have done the work.
So, shown in the upper part of this slide are the people in my laborator
y that contributed to all of the work
I've presented in both Part 1 and Part 2 of my talk.
And in the second part are people at UT Southwestern
that have collaborated...
collaborated with me in analyzing,
especially, the subcellular localization of UBIAD
and these UB... these SCD mice.
And then finally, our funding from the National Institutes of Health.