Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3.
SIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C-terminus of human SIRT3 but lacks an N-terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 5' rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full-length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD(+). Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.
Jin
Lei
L
Sirtris, a GSK Company, 200 Technology Square, Cambridge, MA 02139, USA. ljin@sirtrispharma.com
Galonek
Heidi
H
Israelian
Kristine
K
Choy
Wendy
W
Morrison
Michael
M
Xia
Yu
Y
Wang
Xiaohong
X
Xu
Yihua
Y
Yang
Yuecheng
Y
Smith
Jesse J
JJ
Hoffmann
Ethan
E
Carney
David P
DP
Perni
Robert B
RB
Jirousek
Michael R
MR
Bemis
Jean E
JE
Milne
Jill C
JC
Sinclair
David A
DA
Westphal
Christoph H
CH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009536:Niacinamide / Q000378:metabolism; D021382:Protein Sorting Signals; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D056566:Sirtuin 3; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution / Q000235:genetics*
D006576:Heterocyclic Compounds, 4 or More Rings; D024101:Mitochondrial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C525422:SRT1720; C525046:Sirt3 protein, mouse; D009536:Niacinamide; D056566:Sirtuin 3; D037761:Sirtuins