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The Arf-family, comprised of ADP-ribosylation factors
(Arfs) and Arf-like proteins (Arls,) is a structurally and functionally
conserved group of proteins of approximately 21kDa. Arfs and Arls
are members of the Ras superfamily of regulatory GTP-binding proteins.
Previous research has demonstrated that each Arf isoform has a distinct
function within the cell, including roles in intracellular membrane
traffic and cellular signaling.The GAL4-based two hybrid system
provides a transcriptional assay in vivo in yeast that screens a
human derived cDNA library for novel proteins that interact with
a known bait Arf protein. For this yeast-two hybrid screening of
Arf4Q a human testis library was used. The transformation of the
Arf4Q-BD and Library-AD into CG1945 yeast cells was performed in
a sequential method and tested in a qualitative X-gal assay. Very
few specifics are known about the function of Arf4, but this protein
has been linked to the secretory traffic and golgi morphology functions
within the cell; these cellular processes have also been
implicated in a number of diseases such as Alzheimer's. Results
of the two-hybrid screen will further elucidate the role of Arf
proteins in the cell by defining their interaction with other cellular
proteins and possibly link Arf proteins' cellular function with
a human disorder or disease.
Arf proteins (ADP-ribosylation factors) have been
found to have a number of different activities including the regulation
of membrane traffic in endocytic and exocytic pathways, maintenance
of organelle integrity, assembly of coat proteins and acting as
a cofactor for cholera toxin. The Arf family comprises a group of
structurally and functionally conserved proteins of approximately
21kDa, which are members of the Ras superfamily of regulatory GTP-binding
proteins. The Arf family is divided functionally into three classes
of Arf proteins and ten Arf-like (Arl- proteins). These subfamilies
have arisen by numerous gene duplications. Arfs are distinguished
from Arls by their ability to act as cofactors for cholera-toxin
dependent ADP-ribosylation of the heterotrimeric G protein Gs. Arfs
share more than 60% sequence identity and are highly conserved throughout
evolution. Studies in yeast and cultured mammalian cells indicate
that all the known Arf genes are expressed in all cells. There are
more than a dozen known effectors for Arfs alone and there is mounting
evidence which points to complicated networks of cross reactivity
between Arf family GTPases and effectors; therefore, it is unlikely
that a single activity can appropriately discriminate between the
multiple proteins and allow an unambiguous classification on the
basis of function. Through the use of the yeast two-hybrid assay,
results will hopefully be obtained that will further elucidate the
role of these proteins by defining their interaction with other
proteins.
Data is collected using a GAL4-based two-hybrid system
that provides a transcriptional assay for detecting interaction
in vivo in yeast. This system is used to screen a library for novel
proteins that interact with the known bait protein from the Arf-family.
The bait gene is expressed as a fusion to the GAL4 DNA-Binding Domain
(DNA-BD). While another gene or cDNA from the library is expressed
as a fusion to the GAL4 activation domain (AD). When the bait and
library fusion proteins interact, the DNA-BD and AD are brought
into proximity and activation of transcription of four reporter
genes occurs. [figure 1-Bait Protein: Arf or Arl Protein Library
Protein: from Human Testis Library or Fetal Brain Library The DNA-BD
is AA 1-147 of the yeast GAL4 protein, which binds to the GAL UAS
upstream of the reporter genes. The AD is AA 768-881 of the GAL4
protein and functions as a transcriptional activator. When the Arf-BD
interacts with a library-AD, transcription of the four reporter
genes is activated. One of these reporter genes is the LacZ gene.
When this gene is transcribed it produces galactosidase which cleaves
galactose present in the cell. Yeast colonies with a positive interaction
between the Arf-BD and library-AD will to turn blue when an X-Gal
solution containing galactose is present, creating a qualitative
test for interaction. This assay can be used to identify novel protein
interactions, confirm suspected interactions and define interacting
domains. Use of this method provides immediate access to the genes
encoding the interacting proteins, as well as providing a sensitive
method for detecting relatively weak and transient protein interactions
that may not be biochemically detectable, but critical for proper
functioning in complex biological systems such as the Arf family.
For this yeast-two hybrid screening a new human testis
library was bought. Before the screening was initiated, it was necessary
to establish the library titer and amplify and purify the new library
as well as to determine the the library-AD transformation efficiency
and the appropriate 3AT-concentration needed. The actual transformation
of the Arf-BD and Library-AD was performed in a sequential method
and colonies were picked and transferred onto new plates. Colonies
which displayed adequate growth were streaked and tested in a qualitative
X-gal assay. The yeast plasmid from the positive was extracted,
purified and transformed into E.coli cells. The plasmid was further
purified from these cells and digested and analyzed by agarose gel
electrophoresis.
I. Library Titering The library titer was found to be 3.5 x 10^8
cfu/ml.
II. Amplification and Purificatoin of the Library The testis library
was amplified by plating onto 150mm LB/Amp plates and harvesting
the colonies. Qiagen maxi and gigapreps were used for the purification
of the library plasmid.
III. Finding the Tranformation Efficiency of the Activaton Domain
IV. Determination of Appropriate 3-AT Concentration One of the four
reporter genes transcribed when the Arf-BD and library-AD interact
is the His3 gene. When this gene is turned on additional 3-AT will
suppress its leakiness. The optimal concentration of 3-AT needed
to eliminate background growth but not limit the number of colonies
on -VHis selections plates was determined.
V. Activation and Bait Domain Tranformation A sequential transformation
was used to add both the Arf-BD and library-AD into the yeast strain
CG1945. The testis library-AD was first transformed into the yeast
cells, followed by the transformation with Arf4Q. These cells were
then plated onto SD-Trp-Leu-His + 5mM 3AT. Approximately 100,000
colonies per plate was expected, but the transformation efficiency
of this screen was very low and there were only approximately 150
colonies per plate. A total of approximately 4500 colonies were
screened. From this 500 colonies were picked and patched onto new
plates to allow for further growth.
VI. Colony-Lift Filter Assay Approximately 75 colonies grew well
on the patch plates, and these were streaked onto SD-Leu plates
and then transferred onto a nitrocellulose membrane and exposed
to X-gal. Colonies in which the Arf4Q-BD was interacting with a
library-AD protein demonstrated an activation of transcription of
reporter genes including the LacZ gene causing the colonies to turn
blue with exposure to the X-gal solution. One positive was found
from the Arf4Q screening with the human testis library. This result
is unusual in that the number of potential positives eliminated
by the X-gal assay is extremely high. Normally 80-90% of the clones
tested prove positive with this method, which has been replicated
in further screens using Arl2. Additional testing is being conducted
to determine the reason for the low number of positives in this
screening.
VII. Smash and Grab The positive blue colony was traced back to
the patch plate, picked and grown in selective media. The yeast
plasmid was prepared through the smash and grab procedure and purified
via a qiagen miniprep. This purified plasmid was then transformed
into E.coli cells and plated on LB/Amp plates. LB/Amp media
was innoculated with the resulting colonies which were grown and
harvested. The plasmid was then purified using qiagen miniprep protocol.
VIII. Restriction Digest The purified plasmid DNA was digested and
analyzed by agarose gel electrophoresis to determine the vector
present. The restriction digest showed that the library AD vector
had been isolated not the wanted Arf4Q-BD vector.
The Arf4Q screen using the testis library has yet to provide any
true positives, but additional testing is still being conducted.
The low transformation efficiency in this screen may be due to the
variability in the transformation of yeast. Because of this low
transformation efficiency, the number of colonies that were screened
was very low, limiting the potential number of positives. The use
of a different library may increase the likelihood of finding an
interaction between the Arf4Q-BD and library-AD.
- Continue tests with the purified Arf4Q positive to try to isolate
the Arf-BD vector.
-Send purified plasmid for sequencing to determine the library protein
interacting with Arf4Q
- Yeast-two hybrid screening of Arf4Q and Arf5Q with a human brain
library.
- Arf4Q and Arf5Q have been found to have functions relating to
secretory traffic and golgi morphology which have also been mplicated
in Alzheimer's disease. A yeast-two hybrid screen with the brain
library will possibly link Arf proteins with Alzheimer's.
- Further screening of all Arf and Arl proteins using both human
testis and brain libraries.
- Results of the two-hybrid screen will further elucidate the role
of Arf and Arl proteins in the cell by defining their interaction
with other cellular proteins.
This material is based upon work supported by the Howard Hughes
Medical Institute under Grant No. 52003727 and by the NIH under
grant PO1 HL075209-01. We would also like to acknowledge David Harrison,
Karine Laude, Louise McCann, and Bernard Lassgue for their assistance.
This summer my project centered on a group of proteins found in
the smooth muscle cells in the vasculature called the NADPH oxidase.
This group of proteins come together to form reactive oxygen species
(ROS). ROS has a role in the physiology and function of the vasculature
and examples include superoxide (O2-) and nitric oxide (NO). However,
if there is too much ROS, then vascular disease may arise. One of
these vascular diseases is insulin resistance, a problem where vessels
do not relax optimally to insulin as they should. We have developed
two methods to increase the level of ROS to determine the effect
of ROS on resistance to insulin. We have created a mouse where one
of the proteins in the NADPH oxidase is expressed more often. This
protein is p22phox. If p22phox is expressed more often, then the
NADPH oxidase is more active, leading to more ROS production. A
second method to increase ROS production is through infusing certain
hormones, such as Angiotensin II (Ang II), into mice. Ang II has
the effect of creating more ROS. Therefore, we set up four groups
of mice (mice with overexpression of p22phox and Ang II, mice with
overexpression and no Ang II, mice with no overexpression and Ang
II, and mice with no overexpression and no Ang II). After the groups
had been set up, we treated the mice for fourteen days. At day fourteen,
the mouse was sacrificed, and its aorta was removed. After testing
to see if the vessel was intact, we constricted the vessel and added
doses of insulin to see how much the vessel relaxed. According to
our hypothesis, mice who overexpress p22phox and have Ang II should
relax the least while mice with neither overexpression of p22phox
nor Ang II should have the most relaxation.
insertion of microosmotic pumps into a mouse model, relaxation
studies, data analysis for relaxation studies, Western blotting.
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