Levent DEMiRAY 140104012 Term Paper

REGULATION OF SREBP TRANSCRIPTION FACTOR BY SUMOYLATION AND

ITS EFFECTS ON STEROL REGULATION AND EXPRESSION

Introduction
In the fatty acid and cholesterol biosynthesis, major transcription factors are sterol
regulatory binding proteins (SREBP) which play indispensible role in activation of related
regulatory genes. These transcription factors generally contain a basic helix-loop-helix leucine
zipper (bHLH-zip) motif. SREBP is synthesized on endoplasmic reticulum as a membrane
bound precursor. This is the most distinct difference of SREBP from the other bHLH-zip
transcription factors. The precursor proteins contain two domains: N-terminal transcriptional
activation domain and C-terminal regulatory domain. C-terminal domain interacts with
SREBP cleavage-activating protein (SCAP) which control sterol sensing domain. SREBP-
SCAP complex remain inactive form on ER membrane as long as high level cholesterol
present in cytosol. When cholesterol level gets lower, SREBP-SCAP complexes coated with
ER membrane migrate to the golgi. In golgi, active form of SREBP is released by cleavage of
SREBPs by site-1 and site-2 protease . (1,2)
There are three isoforms(SREBP-1a, SREBP-1c and SREBP-2) present in the cells.
Even though they have different function, they all are subjected to same activation process.
SREBP 1a regulates genes responsible for up taking and synthesizing of cholesterol, fatty
acids, triglycerides, and phospholipids. SREBP2 regulates genes responsible for fatty acid and
sterol biosynthesis. These two isoforms are dominantly found in liver cells.(1)
SREBPs consist of three domain: NH2 terminal domain, hydrophobic transmembrane
spanning part and COOH terminal. DNA binding site is provided by NH2 by dimerization
and H-L-H-LZ motif. It contains 480 aa and rich in serine and proline. COOH is a regulatory
domain containing 590 aa.(1)

Materials

Expression plasmids
-SREBP1 and 2 genes were inserted into pME-GST plasmid in order to obtain pGST SREBP1
and 2 expression plasmids.
-By cloning SUMO(GG and ΔGG) gene into pME-his, pHis SUMO expression plasmid was
obtained.
-By the site directed mutagenesis method, plasmids containing GST tagged SREBP mutant
gene were obtained.
-By inserting SUMO and SREBP genes into pME-RFP and pME-GFP host plasmid, pRFP
SUMO and pGFP-SREBP were constructed.

Cell lines

-HeLa cells were used as a cell line.

- LipofectamineTM was purchased from Invitrogen and used for transfection.

Antibodies

- anti-SUMO antibodies, anti-SREBP1 and 2 polyclonal antibodies were purchased from

Qiagen.

Methods

To begin with, HeLa cells were transfected with pHis SUMO by using the
LipofectamineTM reagent in order for examining sumoylation on SREBP. Then cells were kept
under sterol depleted condition for 48 hours in order to overexpression of SREBPs. After that,
all extracted protein contents of nucleus of cells and the anti-SREBP1 and 2 antibodies were
used for immunoprecipitation and immunoblot analysis. (2)

Following that, in order to determine effect of sumoylation on transcriptional activity

of SREBP-1 and 2, different lines of HeLa cells were transfected with pHis-SUMO(GG) or
pHis- SUMOΔGG expression plasmid. With this way, effect of sumoylation can be identified
by analyzing the differences between the cell transfected with plasmid containing normal
SUMO gene and SUMOΔGG. To do that, SDS-PAGE gel electrophoresis, and
immunoprecipitation, immunoblotting analysis with antibody RS005 and RS004 for SREBP-1
and 2, and antiGMP 1 for SUMO, were done.(fig. 1) (2)
After that, in order to determine effect of sumoylation on amount of SREBP, HeLa
cells transfected with pHis-SUMOΔGG, pGST-SREBP1a and pGST-SREBP2 were grown
under normal conditions and then northern blot analysis on cDNA LDL receptor and HMG
CoA was applied. For controlling the procedure above, it was repeated with pHis-
SUMO(GG) vector instead of pHis-SUMOΔGG.(fig. 2) (2)
In the following step, the aim was to identify sumoylation sites of SREBP. To do that
probable sumoylation sites were determined on the SREBP protein sequence by the help of
Bioinformatic tools. After that, each probable sumoylation site of the SREBP coding gene
was mutated one by one by site directed mutagenesis. Then, sumoylated residues were
identified by immunoblotting with anti-SUMO and SDS-PAGE gel electrophoresis. (fig. 3-4)
 Last of all, fluorescent protein technique was used to identify the location of
sumoylation in the cell. In order to do that, two different vectors were constructed: vectors
containing GFP(Green Fluorescent Protein) tagged SREBP gene and RFP (Red Fluorescent
Protein) tagged SUMO gene. After transfection of HeLa cells with these two vectors, cells
were grown under sterol depleted condition. Finally Delta Vision Core microscope was used
to determine the location.(fig. 5)

Results

Fig 1- Gel picture of immunoprecipitated cell extracts of HeLa cells . (2)

Heavier proteins, due to sumoylation, did not migrate as much as non sumoylated ones
did.

Fig 2-amounts of HMG CoA and LDL receptor in the cells transfected with
SUMO(GG) gene and SUMO(ΔGG). (2)
As it can be seen in figure 2, amount of HMG CoA and LDL receptor of SUMOΔGG
containing cells is lower than SUMO(GG) containing cells. So, downregulation of
sumoylation is revealed.
 Fig 3. the potential sumoylated sites of SREBP(2)

Figure 4- SDS-PAGE gel electrophoresis results after immunoblotting with mutated

SREBPs. (2)
By immunoblotting analysis, residues of SREBP subjected to sumoylation were
determined. In figure 4, there are no Lys123 and Lys 418 band observed on the gel.
Therefore, it can be said that Lys123 and Lys 418 residues are subjected to sumoylation.

Figure 5- Delta Vision Core microscope photograph.(5)

In the photograph, combination of green and red lights

referring to sumoylation can be seen. Red light refers to
SUMO and green refers to SREBP. So, location of
sumoylation is determined as nucleus.
 Discussion

The main purpose of this study was to determine effect of SUMO status on SREBP
and it was proofed that sumoylation is negative regulator of SREBP and so fat metabolism.
Downregulation effect of sumoylation was revealed by determining that amount of HMG
CoA and LDL receptor of SUMOΔGG containing cells is lower than SUMO(GG) containing
cells. Additionally, it was found that lys123 and lys418 residues of SREBP are potentially
subjected to sumoylation by immunoblotting test . Furthermore, location of sumoylation was
determined as a nucleus by using fluorescent protein technology
To sum up, all this findings about sterol and fatty acid metabolism in the terms of
sumoylation can provide us different approaches and cures for diseases caused by problems in
fat metabolism.

References
1) Sato, Ryuichiro. "SREBPs: protein interaction and SREBPs." FEBS Journal. 276.
(2009): 622–627. Print.
2) Hirano, Yuko. “SREBPs are negatively regulated through SUMO-1 modification
independent of the ubiquitin/26S proteasome pathway” The Journal of Biological
Chemistry,
(2003).
3) K. Ilkim, S et. al.; Journal Of Cellular Physiology 191:257–268 (2002)
4) F-P. Zhang,et. Al.; Molecular And Cellular Biology, Sept. 2008, p. 5381–5390
5) Expression and Visualization of Red Fluorescent Protein (RFP) in Neurospora crassa by
Michael Freitag# and Eric U. Selker, Fungal Genetics Newsletter 52:14-17