Isolation and Characterization of Cellular Complexes Containing the Histone Deacetylase SIRT1 Vincent Lew Howard Hughes Medical Institute Fellowship Mentor: Dr. Mark Leid Molecular Pharmacology Lab College of Pharmacy - Oregon State University

DNA   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?

History  Human Jurkat Cells (Immune System)  Silent Information Regulator (SIRT)  Histone DeAcetylase Complex (HDAC)  Chicken (Ovalbumin Upstream Promoter) Transcription Factor Interacting Proteins 1 & 2 (CTIP1 & CTIP2) Represses Gene Transcription

Project Aim  To isolate Silent Information Regulator (SIRT) protein & complexes via purification  To investigate all of the functions of SIRT in the human body  Determine by finding what proteins are found with it

? ? ? SIRT

Activation RNA Polymerase Acetylation Complex RNA Polymerase

Repression CTIP / SIRT Complex

Step One: Phosphocellulose  Phosphocellulose Column  Tightly packed resin  Negatively charged  Wash w/ increasing NaCl molarity (stronger washes) IN COLLECT Na+ Cl - Na+ Cl -

Step Two: Where is the Protein?  Electrophoresis   Negatively Charged & Migrates to Cathode  Smaller strands migrate faster 10% Acrylamide Gel + -

Step Two Continued  Electrotransfer  Use of electricity to transfer proteins from acrylamide gel to nitrocellulose membrane 10% Acrylamide Gel Nitrocellulose Membrane

Step Three: Western Blot  Immunoreactivity  Use of antibodies that bind to specific proteins nitrocellulose membrane chemiluminescence specific protein

Western 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: DEAE  Diethylaminoethyl Column  Tightly packed resin  Positively charged  Wash w/ increasing NaCl molarity (stronger washes) IN COLLECT Na+ Cl - Na+ Cl -

Western 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 Exclusion  Size Exclusion column  Separates based on SIZE of molecules  Use of known markers to determine size of unknown molecules  Thyroglobulin: 669 kD  Catalayse: 232 kD  BSA (albumin): 67 kD  1 kiloDalton = 1.67 x kg

IN OUT Size Exclusion

Western Blot Results Thy (669) Cat (232) BSA (67) Thy (669) Cat (232) 200 mM 400 mM

Step Seven: Immunoprecipitate  Final step of purification in this project  Use of specific antibody (Abx) to bind to individual protein complex  Extract only that which is bound to antibody

Immunoprecipitation Protein X Protein Y SIRT SIRT complexed to unknown protein Sir2 Abx Sepharose Beads

Mass Spectrometry  Determine protein structure based on molecular mass

Results  Still to be determined

Acknowledgements  Howard Hughes Medical Institute  Dr. Mark Leid – Molecular Pharmacology, Oregon State University  Valerie Peterson – Molecular Pharmacology, Oregon State University