Isolation and Characterization of Isolation and Characterization of Cellular Complexes Containing the Cellular Complexes Containing the

Histone Deacetylase SIRT1Histone Deacetylase SIRT1

Vincent LewVincent LewHoward Hughes Medical Institute FellowshipHoward Hughes Medical Institute Fellowship

Mentor: Dr. Mark LeidMentor: Dr. Mark LeidMolecular Pharmacology LabMolecular Pharmacology Lab

College of Pharmacy - Oregon State UniversityCollege of Pharmacy - Oregon State University

DNADNA

Humans have 14,000 to 73,000 micrometers of DNA per chromosome

All 46 chromosomes together in a human cell have 2 meters of DNA which is contained in a nucleus that is 5 micrometers in diameter

How does it fit?

How does it fit?How does it fit?

HistoryHistory

Human Jurkat Cells (Immune System)Human Jurkat Cells (Immune System)

Silent Information Regulator (SIRT)Silent Information Regulator (SIRT)

Histone DeAcetylase Complex (HDAC)Histone DeAcetylase Complex (HDAC)

Chicken (Ovalbumin Upstream Promoter) Transcription Chicken (Ovalbumin Upstream Promoter) Transcription Factor Interacting Proteins 1 & 2 (Factor Interacting Proteins 1 & 2 (CTIP1 & CTIP2CTIP1 & CTIP2))

Represses Gene TranscriptionRepresses Gene Transcription

Project AimProject Aim

To isolate Silent Information Regulator To isolate Silent Information Regulator (SIRT) protein & complexes via purification(SIRT) protein & complexes via purification

To investigate all of the functions of SIRT To investigate all of the functions of SIRT in the human bodyin the human body Determine by finding what proteins are Determine by finding what proteins are

found with itfound with it

?

?

?

SIRT

ActivationActivationRNA

Polymerase

Acetylation Complex

Acetylation Complex

Acetylation Complex

RNA Polymerase

RepressionRepression

RNA Polymerase

CTIP / SIRT Complex

CTIP / SIRT Complex

Step One: PhosphocelluloseStep One: Phosphocellulose

Phosphocellulose Phosphocellulose ColumnColumn Tightly packed resinTightly packed resin Negatively chargedNegatively charged Wash w/ increasing Wash w/ increasing

NaCl molarity NaCl molarity (stronger washes)(stronger washes)

IN

COLLECT

Na+

Cl-

Na+

Cl-

Step Two: Where is the Protein?Step Two: Where is the Protein?

ElectrophoresisElectrophoresis Negatively Charged & Migrates to Cathode Smaller strands migrate faster

10% Acrylamide Gel

+

-

Step Two ContinuedStep Two Continued

ElectrotransferElectrotransfer Use of electricity to transfer proteins from Use of electricity to transfer proteins from

acrylamide gel to nitrocellulose membraneacrylamide gel to nitrocellulose membrane

10% Acrylamide Gel

Nitrocellulose Membrane

Step Three: Western BlotStep Three: Western Blot

ImmunoreactivityImmunoreactivity Use of antibodies that bind to specific proteinsUse of antibodies that bind to specific proteins

nitrocellulose membrane

chemiluminescence

specific protein

Western Blot ResultsWestern Blot Results

Flow Through

300 mM

600 mM

What does this mean?

• There is a significant amount of SIRT protein in the Input & Flow Through

• Input: originally put into the column (before purification)

• Flow Through: protein that did not stick to the column

Input

Step Four: DEAEStep Four: DEAE

Diethylaminoethyl Diethylaminoethyl ColumnColumn Tightly packed resinTightly packed resin Positively chargedPositively charged Wash w/ increasing Wash w/ increasing

NaCl molarity NaCl molarity (stronger washes)(stronger washes)

IN

COLLECT

Na+

Cl-

Na+

Cl-

Western Blot ResultsWestern Blot Results

Input

Flow Through

200 mM

400 mM

600 mM

800 mM

1 M

What does this mean?

• There is SIRT protein in the Input, .2mM & .4mM lanes

• Input: originally put into the column (before purification)

• .2mM: what eluted after .2 milli-Molar NaCl solution

• .4mM: what eluted after .4 milli-Molar NaCl solution

Step Six: Size ExclusionStep Six: Size Exclusion

Size Exclusion columnSize Exclusion column Separates based on Separates based on SIZESIZE of molecules of molecules Use of known markers to determine size of Use of known markers to determine size of

unknown moleculesunknown molecules Thyroglobulin: 669 kDThyroglobulin: 669 kD Catalayse: 232 kDCatalayse: 232 kD BSA (albumin): 67 kDBSA (albumin): 67 kD

1 kiloDalton = 1.67 x 10-24 kg1 kiloDalton = 1.67 x 10-24 kg

IN

OUT

Size ExclusionSize Exclusion

Western Blot ResultsWestern Blot Results

Thy (669)

Cat (232)

BSA (67)

Thy (669)

Cat (232)

200 mM

400 mM

Step Seven: ImmunoprecipitateStep Seven: Immunoprecipitate

Final step of purification in this projectFinal step of purification in this project Use of specific antibody (Abx) to bind to Use of specific antibody (Abx) to bind to

individual protein complexindividual protein complex Extract only that which is bound to antibodyExtract only that which is bound to antibody

ImmunoprecipitationImmunoprecipitation

Protein X

Protein Y

SIRT

SIRT complexed to unknown protein

Sir2 Abx

Sepharose Beads

Mass SpectrometryMass Spectrometry

Determine protein structure based on Determine protein structure based on molecular massmolecular mass

ResultsResults

Still to be determinedStill to be determined

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute

Dr. Mark Leid – Molecular Pharmacology, Dr. Mark Leid – Molecular Pharmacology, Oregon State UniversityOregon State University

Valerie Peterson – Molecular Pharmacology, Valerie Peterson – Molecular Pharmacology, Oregon State UniversityOregon State University