MGC as a Novel Negative Regulator of Cholesterol

1. MGC as a Novel Negative Regulator of Cholesterol

2. Table of Contents:MGC as a novel negative regulator of cholesterol 3 MGC-GST fusion protein project overview (1 week of reading) 3-6 An introduction to the cholesterol signaling pathway 7-8 The discovery of MGC’s negative regulation of cholesterol 9-11 The Cloning of the Mammalian Fusion (4 weeks) 12 Cesium Chloride/Ethidium Bromide DNA Purification (4 days) 13 Calcium Phosphate Transfection into 293t Cells (3 days) 14-15 Confirmation of protein expression with SDS Page Gel/Western Blot (1 day) 16-18 3X SREGFP reporter, and Filipin Stain (4 days) 19 The Cloning of the Bacterial Fusion (3 weeks) 20 Future Directions

3. Project Introduction My project for the semester is the creation of Full-length Mammalian and Bacterial GST Fusion Proteins with a Novel Negative Regulator of Cholesterol (MGC). This involves cloning to specifically ligate MGC “in frame” with GST, confirming the expression of the MGC-GST protein in cells on a SDS Page Gel/Western Blot and demonstrating that the GST MGC protein retains the ability to inhibit cholesterol with a 3X SREGFP reporter and a Filipin Stain. These proteins will be used to study protein-protein interactions with MGC (i.e.-a sterol regulator of SCAP and Insig-1) and to generate an MGC antibody respectively.

4. Cholesterol Signaling Cholesterol is a major component in animal cell membranes and cells employ a family of membrane-bound proteins, who, through a feedback system, monitor its concentration and adjust its synthesis and uptake. Insig retains SREBPs (and SCAP) in the ER

5. Sterol Regulatory Elements Mediate Cholesterol DependantRegulation of Transcription

6. Cholesterol Signaling cont’d SREBPS are precursors in the ER that are cleaved before entering the nucleus

8. MGC as a Novel Negative Regulator of Cholesterol Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells Monika Suchanek, Anna Radizowska & Christoph Thiele Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Published online March 23, 2005 The PGRMC protein with a similar domain cytochrome B5 heme-binding/progesterone receptor (in human Hpr6) was found to bind to the sterol regulatory protein Insig-1. Hpr6 (Heme-1 Domain Protein) Regulates the susceptibility of Cancer Cells to Chemotherapeutic Drugs Gerard Crudden, Rachel. Chitti, and Rolf J. Craven Department of Molecular and Biomedical Pharmacology, Markey Cancer Center, University of Kentucy, Lexington, Kentucky Received August 25, 2005; accepted October 14, 2005 PhD Candidate student Anthony Bruce verified through a co-immunoprecipitation in 2006 that the protein MGC interacts with the Insig-1 protein.

9. A further experiment by research associate Dr. Xinchang Feng showed that MGC conferred the stability of Insig-1 when treated with Cycloheximide, an inhibitor of protein biosynthesis. MGC stabilizes Insig1 in 293 T cells Time in Min of Cyclohexamide Treatment 120 60 30 0 120 60 30 0 Actin Myc-Insig1 M2 Flag (MGC) EV + Insig1 MGC + Insig1

10. pcDNA3 pcDNA3-N-GST

11. The Cloning of the Mammalian Fusion 1. The parental plasmid, pcDNA3-N-GST was sequentially digested with ApaI and HindIII. 2. The MGC-c-flag cDNA was sequentially digested out using ApaI and HindIII from its parental pcDNA3.0. The excised inserts were gel extracted and purified used a MO BIO kit. 3. Ligation of the ApaI/HindIII MGC-c-flag inserts into the ApaI/HindIII pcDNA3-N-GST was completed using a MO BIO kit. 1% Agarose Gel of MGC-c-flag excised with HindIII and ApaI 1 2 3 4 5 6 7 8 9 10 11 12 13 14

12. Cesium Chloride/Ethidium Bromide Purification Method For Large Scale preparations of DNA, the Cesium Chloride/Ethidium Bromide purification method is used. • The first steps involved the Alkaline Lysis Method, a series of centrifugations, re-suspensions and washes with ethanol. • CsCl and EtBr solutions were added to the DNA and the tops of the 20ml tubes were quick-sealed. The tubes were spun at 60,000rpm for 24 hours at 20 °C • The tubes were pierced and the bands were extracted with a needle. After each extraction, a volume of H20-saturated butanol was added. This was repeated until until no EtBr remained and the solution was clear 4. Three volumes of water were added to the remaining DNA and it was washed with EtOH twice to dissolve the pellet.

13. Calcium Phosphate Transfection(In Human Embryonic Kidney (HEK) 293T cells) 1. Calcium Chloride was added dropwise with DNA to a HBSP phosphate-buffered solution, creating nano-complexes. 2. Nano-complexes were introduced to 70-80% confluent cells, and were uptaken via endocytosis. 3. The Cell media was changed 12 hours after the transfection.

14. SDS Page Gel/Western Blot 1. Proteins were boiled with SDS which caused denaturation and applied an overall negative charge based on the composition of the individual amino acids that make up the protein 2. A Nitrocellulose membrane was placed on the gel 3. Electrophoresis was used to transfer the protein bands to the nitrocellulose membrane. The nitrocellulose membrane is imprinted with the same protein bands as the gel. Audioradiography The membrane was probed with a primary, Anti-GST, then a secondary antibody HRP conjugate (ECL chemiluminescent reaction). The blot was developed in the darkroom. The enzyme Horseradish Peroxidase

15. 1 2 3 4 5 Western Blot of MGC-GST fusion protein, expressed in pcDNA3 transfection to 293T cells. 47.5KD Anti-GST 32KD 1: MGC-GST 2: MGC-GST 3: Delta-C-GST 4. Delta-N-GST 5: Delta-N-GST MW MGC 28 MW GST 25

16. MGC downregulates SREBP Pathway 10ugEV + SREGFP 10ugMGC + SREGFP

17. 3X SRE GFP Reporter 1.DeltaNMGCGST2cSREGFP 2. EVGSTSREGFP 3. MGCGST1cSREGFP 4. MGCGST2cSREGFP 1 2 3 4

18. Filipin Staining The fluorescent probe filipin has been used to detect unesterified cholesterol. (5% regular media) MGC EV With the Filipin staining, the MGC stains showed less intercellular cholesterol, thus, the function of MGC was shown to be retained.

19. The Cloning of the Bacterial Fusion • Sequential double digests with BamHI and SmaI successfully digested the parental pGEX4T.1 vector • 2. A 1% DNA Agarose Gel was run and the linearized pGEX4T.1 was gel extraction purified with a MO BIO Kit • 3. MGC-C-terminal and MGC-N-terminal were excised by sequential ApaI and BamHI digests. After the ApaI digest, the linearized fragments were blunt-ended with a Pfu end-filling reaction, prior to BamHI digestion. • 4. The C-terminal and N-terminal fragments were run on a 1% DNA Agarose Gel and were gel extraction purified using a MO BIO kit (see figure). • 5. Ligation of MGC-C-terminal and MGC-N-terminal ApaI/Pfu/BamHI purified fragments into pGEX4t.1 SmaI/BamHI plasmid was performed. Ligations were transformed into bacteria

20. Successful Cloning of MGC Deletion Mutants into pGEX41.1 (GST Plasmid) Bacterial fusion cut with BamHI and ZhoI C-terminus N-Terminus

21. Future Directions • Mammalian Fusion • Completion of a GST fusion assay to prove function of MGC-GST protein • Bacterial Fusion • The Bacterial GST-MGC fusion will be used to create an MGC antibody