SIRT1 shows no substrate specificity in vitro.
SIR2 is a key regulator of the aging process in many model organisms. The human ortholog SIRT1 plays a pivotal role in the regulation of cellular differentiation, metabolism, cell cycle, and apoptosis. SIRT1 is an NAD(+)-dependent deacetylase, and its enzymatic activity may be regulated by cellular energy. There is a growing number of known SIRT1 substrates that contain epsilon-acetyl lysine but for which no obvious consensus sequence has been defined. In this study, we developed a novel unbiased method to identify deacetylase sequence specificity using oriented peptide libraries containing acetylated lysine. Following incubation with SIRT1, the subset of deacetylated peptides was selectively captured using a photocleavable N-hydroxysuccinimide (NHS)-biotin linker and streptavidin beads and analyzed using mass spectrometry and Edman degradation. These studies revealed that substrate recognition by SIRT1 does not depend on the amino acid sequence proximate to the acetylated lysine. This result brings us one step closer to understanding how SIRT1 and possibly other protein deacetylases chose their substrate.
Blander
Gil
G
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Olejnik
Jerzy
J
Krzymanska-Olejnik
Edyta
E
McDonagh
Thomas
T
Haigis
Marcia
M
Yaffe
Michael B
MB
Guarente
Leonard
L
D000595:Amino Acid Sequence; D017209:Apoptosis; D001671:Biochemistry / Q000379:methods; D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D006655:Histone Deacetylases / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D056564:Sirtuin 1; D037761:Sirtuins / Q000737:chemistry / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry; D013379:Substrate Specificity; D013388:Succinimides / Q000737:chemistry
D019151:Peptide Library; D010455:Peptides; D013388:Succinimides; D001710:Biotin; D019809:Streptavidin; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D037761:Sirtuins; D006655:Histone Deacetylases; D008239:Lysine; C001426:N-hydroxysuccinimide