MGC as a Novel Negative MGC as a Novel Negative Regulator of CholesterolRegulator of Cholesterol

Table of Contents:Table of Contents:MGC as a novel negative regulator of cholesterolMGC as a novel negative regulator of cholesterol

3 3 MGC-GST fusion protein project overview (1 week of reading)MGC-GST fusion protein project overview (1 week of reading)3-6 3-6 An introduction to the cholesterol signaling pathwayAn introduction to the cholesterol signaling pathway7-87-8 The discovery of MGC’s negative regulation of cholesterolThe discovery of MGC’s negative regulation of cholesterol9-119-11 The Cloning of the Mammalian Fusion (4 weeks)The Cloning of the Mammalian Fusion (4 weeks)1212 Cesium Chloride/Ethidium Bromide DNA Purification (4 days)Cesium Chloride/Ethidium Bromide DNA Purification (4 days)1313 Calcium Phosphate Transfection into 293t Cells (3 days)Calcium Phosphate Transfection into 293t Cells (3 days)14-1514-15 Confirmation of protein expression with SDS Page Gel/Western Blot Confirmation of protein expression with SDS Page Gel/Western Blot

(1 day)(1 day)16-1816-18 3X SREGFP reporter, and Filipin Stain (4 days) 3X SREGFP reporter, and Filipin Stain (4 days)19 19 The Cloning of the Bacterial Fusion (3 weeks)The Cloning of the Bacterial Fusion (3 weeks)2020 Future DirectionsFuture Directions

My project for the semester is the creation of Full-length Mammalian and Bacterial GST 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 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 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-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 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 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.sterol regulator of SCAP and Insig-1) and to generate an MGC antibody respectively.

Project Introduction

Cholesterol SignalingCholesterol SignalingCholesterol 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

Sterol Regulatory Elements Mediate Cholesterol DependantSterol Regulatory Elements Mediate Cholesterol DependantRegulation of TranscriptionRegulation of Transcription

Cholesterol Signaling cont’dCholesterol Signaling cont’d

SREBPS are precursors in the ER that are cleaved before entering the nucleus

MGC as a Novel Negative Regulator of CholesterolMGC as a Novel Negative Regulator of CholesterolThe PGRMC protein with a similar domain cytochrome B5 heme-binding/progesteroneThe PGRMC protein with a similar domain cytochrome B5 heme-binding/progesteronereceptor (in human Hpr6) was found to bind to the sterol regulatory protein Insig-1. receptor (in human Hpr6) was found to bind to the sterol regulatory protein Insig-1.

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

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 PhD Candidate student Anthony Bruce verified through a co-immunoprecipitation in 2006 that the protein MGC interacts with the Insig-1 protein. that the protein MGC interacts with the Insig-1 protein.

EV + Insig1 MGC + Insig1

Time in Min ofCyclohexamideTreatment

0 030 3060 60120 120

M2 Flag (MGC)

Myc-Insig1

Actin

MGC stabilizes Insig1 in 293 T cells

A further experiment by research associate Dr. Xinchang Feng showed that MGC 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 conferred the stability of Insig-1 when treated with Cycloheximide, an inhibitor of protein biosynthesis.protein biosynthesis.

pcDNA3

pcDNA3-N-GST

The Cloning of the Mammalian FusionThe Cloning of the Mammalian Fusion

1. The parental plasmid, pcDNA3-N-GST was sequentially digested with ApaI and 1. The parental plasmid, pcDNA3-N-GST was sequentially digested with ApaI and HindIII. HindIII.

2. The MGC-c-flag cDNA was sequentially digested out using ApaI and HindIII from 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 its parental pcDNA3.0. The excised inserts were gel extracted and purified used a MO BIO kit. MO BIO kit.

3. Ligation of the ApaI/HindIII MGC-c-flag inserts into the ApaI/HindIII pcDNA3-N-3. Ligation of the ApaI/HindIII MGC-c-flag inserts into the ApaI/HindIII pcDNA3-N-GST was completed using a MO BIO kit.GST was completed using a MO BIO kit.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1% Agarose Gel of MGC-c-flag excised with HindIII and ApaI1% Agarose Gel of MGC-c-flag excised with HindIII and ApaI

Cesium Chloride/Ethidium Bromide Cesium Chloride/Ethidium Bromide Purification MethodPurification Method

For Large Scale preparations of DNA, the Cesium Chloride/Ethidium Bromide For Large Scale preparations of DNA, the Cesium Chloride/Ethidium Bromide purification method is usedpurification method is used. .

1.1. The first steps involved the Alkaline Lysis Method, a series of centrifugations, The first steps involved the Alkaline Lysis Method, a series of centrifugations, re-suspensions and washes with ethanol. re-suspensions and washes with ethanol.

2.2. CsCl and EtBr solutions were added to the DNA and the tops of the 20ml tubes 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 were quick-sealed. The tubes were spun at 60,000rpm for 24 hours at 20 °C°C

3.3. The tubes were pierced and the bands were extracted with a needle. After each The tubes were pierced and the bands were extracted with a needle. After each extraction, a volume of Hextraction, a volume of H220-saturated butanol was added. This was repeated 0-saturated butanol was added. This was repeated

until until no EtBr remained and the solution was clearuntil until no EtBr remained and the solution was clear

4.4. Three volumes of water were added to the remaining DNA and it was washed Three volumes of water were added to the remaining DNA and it was washed with EtOH twice to dissolve the pellet.with EtOH twice to dissolve the pellet.

Calcium Phosphate TransfectionCalcium Phosphate Transfection(In Human Embryonic Kidney (HEK) 293T cells)(In Human Embryonic Kidney (HEK) 293T cells)

1. Calcium Chloride was added dropwise with DNA to a HBSP phosphate-buffered 1. Calcium Chloride was added dropwise with DNA to a HBSP phosphate-buffered solution, creating nano-complexes. solution, creating nano-complexes.

2. Nano-complexes were introduced to 70-80% confluent cells, and were uptaken 2. Nano-complexes were introduced to 70-80% confluent cells, and were uptaken via endocytosis. via endocytosis.

3. The Cell media was changed 12 hours after the transfection.3. The Cell media was changed 12 hours after the transfection.

SDS Page Gel/Western BlotSDS Page Gel/Western Blot1. 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 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 proteinthe protein2. A Nitrocellulose membrane was placed on the gel 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.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.

AudioradiographyAudioradiographyThe membrane was probed with a primary, Anti-GST, then a secondary The membrane was probed with a primary, Anti-GST, then a secondary antibody HRP conjugate (ECL chemiluminescent reaction). The blot was antibody HRP conjugate (ECL chemiluminescent reaction). The blot was developed in the darkroom.developed in the darkroom.

The enzyme Horseradish Peroxidase

Western Blot of MGC-GST fusion protein, expressed in pcDNA3 transfection to 293T Western Blot of MGC-GST fusion protein, expressed in pcDNA3 transfection to 293T cells.cells.

Anti-GST

1 2 3 4 5

1: MGC-GST

2: MGC-GST

3: Delta-C-GST

4. Delta-N-GST

5: Delta-N-GST

47.5KD

32KD

MW MGC 28

MW GST 25

MGC downregulates SREBP MGC downregulates SREBP PathwayPathway

10ugEV + SREGFP 10ugMGC + SREGFP

3X SRE GFP Reporter3X SRE GFP Reporter 1.DeltaNMGCGST2cSREGFP

2. EVGSTSREGFP

3. MGCGST1cSREGFP

4. MGCGST2cSREGFP1 2

3 4

Filipin StainingFilipin Staining

EV MGC

The fluorescent probe filipin has been used to detect unesterified cholesterol. (5% regular media)

With the Filipin staining, the MGC stains showed less intercellular cholesterol, thus, the function of MGC was shown to be retained.

The Cloning of the Bacterial Fusion The Cloning of the Bacterial Fusion

1.1. Sequential double digests with BamHI and SmaI Sequential double digests with BamHI and SmaI

successfully digested the parental pGEX4T.1 vectorsuccessfully 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

Successful Cloning of MGC Deletion Mutants into pGEX41.1 Successful Cloning of MGC Deletion Mutants into pGEX41.1 (GST Plasmid)(GST Plasmid)

C-terminus

N-Terminus

Bacterial fusion cut with BamHI and ZhoI

Future Directions Future Directions

Mammalian FusionCompletion 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