Research Proposal- Miri Hillel 043468446:
Modeling Ufd4 in S.cerevisiae as an E3-ligase and/ or a chaperone in the Ubiquitin- Proteasome
System.
I.Specific aims of the research:
The Ubiquitin-Proteasome System (UPS) within the eukaryotic cell is a proteolytic system in
which aberrant or misassembled proteins are degraded via Ub-ligase (E3) enzymes. One
pathway within the UPS is Ub-fusion degradation (UFD). Here, we focus on Ufd4, an 1483AA,
168kDa E3 ligase of this pathway in S.cerevisiae, and two of it’s substrates: Ubc7 and UBB+1.
Ubc7 is an ER membrane-associated E2 enzyme that is normally degraded by Ufd4 when found
in the cytosol. However, Ubc7 is not degraded when Ufd4 is mutated and it accumulates in
cytosolic aggregates [1]. UBB+1 is a mutated form of human Ubiquitin B. It’s aberrant 19AA
longer tale is an outcome of molecular misreading causes loss of original stop codon. It
accumulates in neurons in neurodegenerative diseases and in inclusion body myositis. It is
presented in higher amounts when cells go through stress and this accumulation leads to a
general block of the proteasome, while mutated UBB+1 that cannot be ubiquitilated does not [2].
Our long-term goal is to evaluate the properties and function of Ufd4 and to clarify the
differences (if there are any) between mediating degradation of endogenous substrates and
toxic mutants. We examine this issue using Ubc7 as a endogneous model substrate and UBB+1
as a toxic model substrate. Regarding Ubc7, our hypothesis is that Ufd4 acts either as a polyUb chain elongating factor or as a chaperone escorting poly-ub Ubc7 to the proteasome. We
also hypothesize that the N-terminus of Ufd4 is essential for the interaction with the proteasome.
As for UBB+1, we assume that preventing its ubiquitilation by diminishing Ufd4, will impair
proteasomal inhibition, as known for unubiquitilated UBB+1. We also think that elongating the Cterminal tale of UBB+1 or making it more hydrophobic will make it more easily degraded.
Clarifying the mechanism of function of Ufd4 may elucidate the mechanism of E3-ligases in
general and may also help us better understand E3-associated diseases like Angelman
syndrome and Fanconi anemia. Revealing the specific role of Ufd4 in degradation of UBB+1 may
help us progress with understanding how to prevent proteasomal inhibition in neurons of AD
patients, and as a result, to postpone, or even prevent, neuronal death.
We will pursue this study in two specific aims:
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Ubc7 as an endogenous model substrate: Ufd4 elongating/ escorting function will be
tested using mutants of Ufd4 and checking the maximal length of poly-Ub chains attached
on tagged Ubc7 compared to WT. to explore whether Ufd4 is a chaperon we’ll use Ufd4
mutants compared to WT cells and check in vivo for Ubc7 rate of degradation via
cycloheximide chase. We expect to find that Ufd4 is both elongating factor and a
chaperone.
UBB+1 as a toxic model substrate: First we will have to ensure mutated Ufd4s do not
interact with UBB+1 using an IP assay. Then, UBB+1 (with different tale lengths) rate of
degradation will be tested through cycloheximide chase assay in WT and mutants of Ufd4 in
vivo. Since UBB+1 accumulates in stress, we will also perform a growth test of WT cells and
ufd4 mutants with UBB+1consisting different tale lenghts on selective plates w/o Canavanine
for stress induction.
II. Significance:
For all known by now, cellular quality control of protein synthesis happens in the ER where
proteins go through various modifications. Unfolded or misassembled proteins are degraded via
Ub-proteasome system. Here we wish to prove that Ufd4 functions as a quality control factor in
the cytosol when degrading misslocalized Ubc7Showing protein quality control happens also in
the cytosol can open a whole new research branch. And if so, could quality control happen in
the nucleus as well? Confirming quality control happens not only in the ER but also in the
cytosol (and who knows where else…)can significantly influence cellular biology researches.this
might change everything we think we know about quality control and how cells deal with
unfolded proteins.
UBB+1 is highly presented in neurodegenerative damaged neurons. For all that known by now,
UBB+1 is not the cause for these diseases, but it’s accumulation is definitely worsen neuronal
condition by enabling proteasomal inhibition. Once proteasome is inhibited, the cell cannot
degrade harmful proteins and they aggregate in the cell and poisoning it. Enabling degradation
of UBB+1 could help elongating cell life. It is highly important for us to crack UBB+1 mystery since
it could lead a progress in neurodegenerative diseases research field and will allow researchers
to expand their research tools.
III. Innovation:
This research sets a goal to fully define the properties of Ufd4 as an E3-ligase by combined
strategy in which Ufd4 function is being tested not only with endogenous substrate but also with
toxic mutant.
We want to explore the properties of Ubc7 inclusions received in Ufd4ΔCue1Δ cells. To do this
we will use inclusions markers to check whether Ubc7 forms Juxtanuclear Quality Control
inclusions (JUNQ) which are known for having contents of still processed materials and
misfolded proteins. Or maybe Ubc7 forms Insoluble Protein Deposit (IPOD) inclusions which are
known for having contents of materials that cannot be used by the cell and are even toxic[3].
This is the first time we will use this kind of approach and it will also be a first time in which Ubc7
inclusions are characterized.
As for UBB+1, it is unusual to assume that endogenous common pathway like ubiquitilation can
sometimes be harmful. Here we suggest that arresting this pathway might save the cell from
proteasomal inhibition and allow the cells exist with UBB+1 aggregates in it.
IV. Research approaches:
1. Ubc7 as an endogenous model substrate:
In order to identify the specific role of Ufd4 we will use WT and Ufd4 mutated yeast. The
mutations we use are well known mutations which damage the function of Ufd4 in a way or
other:
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Ufd4∆- Complete lack of Ufd4.
Ufd4∆N- Ufd4 lacking it’s first 201 AA.
Ufd4 C1450S- Catalytic Cystein in position 1450 was replaced to Serine.
Ufd4 IL298-299AA- Armadillo repeat site elongating/ escorting function will be tested
using mutants of Ufd4 and checking the maximal length of poly-Ub chains attached on
tagged Ubc7 compared to WT.
(All strains, including WT, have HA- tagged Ubc7 and identical genomic background but
the specific mutation)
These strains will allow us to perform a few different assays which are about to help us
explore the interaction properties of Ufd4 and Ubc7 from different aspects:
A. Ufd4-dependent degradation rate of Ubc7 assay:
In order to estimate the rate Ufd4-dependent degradation conditions of ubc7, we will
perform a Cycloheximide chase assay. In this assay 1O.D/ml of cells are harvested from
liquid media and Cyloheximide is added. Time samples are taken and cell lysis is
performed. 0.25 O.D of each sample is then loaded into 5-15% gradient SDS gel. After
gel transfer, membrane is exposed to the proper antibodies.
During this assay, there are many elements which can go wrong but are fixable. One of
them is finding there is no protein expression in the cells. To avoid this frustrating
situation, we will first demonstrate a steady state assay to cells to make sure Ubc7 is
expressed: In case we find Ubc7, we will have to check the quality of our antibodies with
other proteins or to change gel properties. In case Ubc7 is not to be seen in steady state
assay, we will have to go back to molecular biology, since HA-Ubc7 is endogenously
expressed and is not by plasmid. Another possible problem is seeing no degradation in
WT on membrane. This could happen either if the WT strain is not really WT, which can
be solved by sequencing, or if the Cycloheximide is not good for some reason, then we
will have to use a fresh tested Cycloheximide solution. To make sure transfer worked
well, we will expose the membrane to ponceau stain pre western blot to see if there are
any proteins on the membrane.
Success in this assay will be detecting significant changes in degradation rate of Ubc7
between cells expressing Ufd4 and cells expressing it’s mutants.
B. Ubc7-Ura expressing cells growth test:
For this assay we will use specific yeast strain- knocked out for His, Ura and Trp and is
also Ufd4∆ Ubc7∆ and Cue1∆ (Cue1 anchors Ubc7 to the ER). These cells will go
through co- transformation with Ubc7-Ura fusion encoding plasmid under Trp selection
and one of the Ufd4 mutants plasmid under His selection. The hypothesis is once Ubc7Ura is degraded by Ufd4 or one of it’s mutants, growth on –Ura media will be impaired.
After co-transformation onto a –His-Trp media, samples will be taken from each clone to
–His-Trp-Ura plate growth test. Our negative control will be the primary strain with no
transformations or only one transformation while our positive control will be the same
strain transformed with three selective empty vectors (His, Trp & Ura).
One major cause which could divert the results is the fact that our Ubc7-Ura fusion
protein will be over-expressed under this episomal plasmid and not in endogenous level.
We will perform a western blot to compare levels of expression/ degradation of overexpressed Ubc7 Vs. endogenous Ubc7. If major differences will occur, then we will have
to insert our fusion protein to the cells via integrative plasmid and repeat the experiment:
transform Ufd4 or it’s mutants and perform another growth test.
We hope this assay will allow us to reveal a little more about the contribution of each
Ufd4 mutant to stabilization of Ubc7 in the cells.
C. Ubc7 localization assay:
We already know Ubc7 is enchored to the ER membrane via Cue1. When fusing Ubc7
with GFP, indeed we see beautiful ER staining. Alternatively, when Cue1 is diminished,
GFP-Ubc7 is nowhere to be seen since Ufd4 leads to it’s degradation. When removing
Ufd4 as well, we can see cytosolic staining which turns into what looks like aggregates in
Cue1∆Ufd4∆ cells. We plan to use aggregation markers to identify which type of
inclusions does Ubc7 creates, JUNQ or IPOD. We will transform our double knock-out
cell with each of the markers and see which of them forms fused inclusions with Ubc7. A
merge of Ubc7 aggregates with JUNQ marker means Ubc7 forms inclusions in which the
cell is still processing their contents. In contrast, if Ubc7 inclusions will fuse with IPOD
marker, it will mean these inclusions are toxic and considered as waste that cannot be
further processed in benefit of the cell. In case we will find Ubc7 fused with both kind of
markers, we will have to estimate the quantity of Ubc7 merge with each marker for
statistical use. We will then try to restore expression of Ufd4 to see what alters in the
aggregates and with Ubc7 localization.
This assay is in process these days and many hours are spent with fluorescent
microscope in a dark room. We were recently able to see about 50% aggregated cells of
all cells in the double knock-out cells. We think our population of double knock-out cells
has got infected and we are nowadays trying to isolate aggregated cells. In parallel, we
want to perform sequence analysis to both populations to make sure there is more than
one strain on our plate, so we will be positive all our double knock-out cells perform
aggregates and not only half of them.
If we will be able to prove the aggregates forms as a cause of relocation of Ubc7 from
the ER to the cytosol and due to the fact Ufd4 is missing, it will be major evidence that
Ubc7 degradation relies on one pathway only, UFD pathway via Ufd4. We would also
like to be able to restore Ubc7 degradation after re-expression of Ufd4 to strengthen our
hypothesis.
D. Ubc7 and Ufd4 proteasome interaction assay:
It is known that Ufd4 is interacting with Rpt4 and Rpt6 subunits of the proteasome. But
do Ubc7 interacts with those subunits too? And if so, does interaction of Ubc7 with the
proteasome is Ufd4-dependent, meaning they form a tertiary complex? To answer these
questions we are about to test the interaction of these two proteins with the proteasome.
This assay will be performed In vitro via affinity column. We are still discussing the
protocol and therefore there isn’t yet much to say about it right now.
E. Ufd4 random mutagenesis:
We will perform random mutagenesis of Ufd4 looking for more mutations inhibiting
catalytic activity/ interaction of Ufd4 with Ubc7. Once these mutations are found, we will
perform a scan to help us understand the consequences of each mutation on
degradation rate of Ubc7 using the same system from paragraph B: Ufd4∆Ubc7∆Cue1∆
cells transformed with Ubc7-Ura and a random mutagenesis product. We will perform
this assay on a 96-well plate in the TECAN scanner. The analysis will run for 24-48
hours and by the end of it we should know if there are any other important sites on Ufd4
which are required for Ubc7 degradation. All mutations found will go through sequencing
and through all assays mentioned above to see which is functional inhibitor and which is
interaction inhibitor.
2. UBB+1 as a toxic model substrate:
Ufd4 is part of UFD pathway which degrades also Ubiquitin B. we want to know if Ufd4 is
capable to initiate degradation of UBB+1, a toxic human substrate found in neurodegenerative-damaged neurons.
A. Ufd4 mutants non-interaction with UBB+1 IP assay:
We will have to make sure our toxic substrate does not interact with Ufd4 mutants
and this will be made by IP assay. Cell lysate will be pulled down with α- UBB+1
antibodies to see if there are ant Ufd4 mutants that are still capable to attach the
substrate.
We will have to make sure the antibody we are using is UBB+1 specific and cannot
parcipitate Ubiquitin B. we would like to see that no Ufd mutant can interact with
UBB+1. In we do realize one (or more) of our mutant do interact with UBB+1, we will to
fint their interaction site and to mutate it. The reason we do it is because our known
mutants are bearing Ufd4 functional mutations and but we are not sure these
mutations can abolish all Ufd4 interactions. Therefore we are still looking for
interaction mutations, like mentioned in paragraph 1E.
B. different length and hydrophbicity UBB+1 degradation assay:
Previous studies have shown that UBB+1, a 19 AA longer protein than Ubiquitin B,
could not degrade and could even inhibit the proteasome. It has been also shown
that longer (than 19 AA) tailed UBB+1 does not accumulate in the cell, does not
inhibit the proteasome and is normally degraded. We also now that ubiquitilated
UBB+1 is the cause for proteasom inhibition. We would like to express UBB+1 with
different tail lengths and hydrophobicity in the cells and perform Cycloheximide
assay to see which mutant is most stable. In order to check the effect of UBB+1
ubiquitilation on proteasomal inhibition we will express different UBB+1 mutants with
different Ufd4 mutants.
We will repeat this experiment with tail as long as mentioned above with different
hydrophobicity levels and then we will compare the degradation rates. Our success
in this assay will be to see a significant change in degradation rates of UBB+1 with
different tail lengths/tail hydrophobicity. We believe the longer/more hydtophobic the
tail is, the better UBB+1 degradation is. We would like to see significant changes of
UBB+1 degradation with mutated Ufd4 and which mutant is the most stabilizing factor
of UBB+1. We will be happy to find that UBB+1 best degradation received in two
cases: first- when UBB+1 has longer than 19 AA/hydrophobic tail. This kind of protein
should be harmless to the cell and should degrade easily in the presence of Ufd4.
Second- in the absence of Ufd4 when UBB+1 has it’s regular 19AA tail. Since
ubiquitilation of UBB+1 is the proteasomal inhibiting factor, we believe expelling Ufd4
might reduce UBB+1 ubiquitilation and thus prevent inhibition of such an important
cellular element like the proteasome. In case we will see that diminishing Ufd4 has
no positive influence on the cells (meaning, the proteasome is still inhibited) we will
have to check again a few things: we will have to make sure UBB+1 is not
ubiquitilated via another E3- ligases but Ufd4. If we do find an alternative
ubiquitilation pathway for UBB+1 we will have to knock-out the cell for the other E3ligase.
C. Cells growth test with Canavanine stress:
It has been shown before that UBB+1 expressing cells can manage the presence of
UBB+1 and degrade it while it’s endogenous level are low. Once a cell stress occurs
and UBB+1 level go a little higher, it is getting more and more difficult to the cell to
degrade UBB+1, it accumulates in the cells, aggregates are formed and pretty soon
afterwards the cell goes through apoptosis. We want to see how significant exactly
stress induction is to UBB+1 expressing with Ufd4 mutants cells vitality.
To strength the results received in this assay we will run another Cycloheximid assay
with Canavanine stressed cells.
We hope to see stabilization in cells with longer tale and Ufd4 and in cells with UBB+1
with 19AA tail and no Ufd4 from the same reason mentioned in paragraph 2B. In
case we will not get the desirable results we will use the same strategies mentioned
in paragraph 2B.
V. bibliography list:
1. Ravid, T. and M. Hochstrasser, Autoregulation of an E2 enzyme by ubiquitin-
chain assembly on its catalytic residue. Nat Cell Biol, 2007. 9(4): p. 422-7.
2. van Leeuwen, F.W., et al., Molecular misreading: the occurrence of frameshift
proteins in different diseases. Biochem Soc Trans, 2006. 34(Pt 5): p. 738-42.
3. Kaganovich, D., R. Kopito, and J. Frydman, Misfolded proteins partition
between two distinct quality control compartments. Nature, 2008. 454(7208):
p. 1088-95.