Thousands of researchers around the world are studying the connections between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlights how Abcam's MitoSciences range of research tools has contributed to this effort. Read the full list of 37 original research papers published this month.
Past issues are available for review in the archives.
STACs (Sirtuin activating compounds)
Sirtuins are a family of NAD+ dependant enzymes that act as deacetylases or ADP-ribosyl transferases to regulate metabolic pathways. They depend on NAD+ so their activity is directly linked to mitochondria and the energy status of the cell. The effect of deacetylation or ADP-ribosyl transfer is primarily of protection against age related diseases including cancer, inflammation, cardiovascular disease, diabetes, neurodegeneration, and aging. Undoubtedly, activation of these enzymes has enormous therapeutic potential.
One way SIRT1 promotes cell survival is by negatively regulating p53. In 2003, the lab of David Sinclair identified a number of plant polyphenols, including resveratrol that, in an in vitro assay, activated SIRT1 dependant deactylation of a fluorophore tagged p53-K382 acetylated peptide. Subsequently in yeast these sirtuin activating compounds (STACs) also led to the deactylation of p53 K382 and an increase in lifespan.
However several groups were unable to reproduce these findings when using non-fluorophore tagged peptide substrates. This suggested that the fluorophore itself had played a role in the sirtuin activation assay. Questions arose whether STACs did indeed function as direct activators of SIRT1. With this uncertainty the ongoing clinical trials and the potential therapeutic benefit of STACs has become controversial in recent years.
Two papers this month provide interesting new evidence in support of the original work and hypothesis. The Sinclair lab evaluated whether the STACs bound only the fluorescent tag in the original assay or whether the hydrophobic fluorophore mimicked the properties of natural substrates. These researchers created assays measuring deactylation by enzymatic and mass spectrometry means to eliminate the need for fluorescence reporting. First they determined that when a bulky hydrophobic fluorescent tag was present it only facilitated STAC induced activation of the deactylase activity when placed in specific positions in the peptide sequence relative to the acetylated lysine and that the fluorophore was not necessary for activation but could be replaced by bulky hydrophobic amino acids at +1 and +6 positions.
Therefore the assay used in the original work had fortuitously placed a hydrophobic moiety in a position necessary to allow STACs to activate the deactylase activity of SIRT1. Next using fluorophore free acetylated peptide sequences from the mitochondrial biogenesis transcription factors, PGC1a and FOXO3a, these researchers showed that deactylase activation also occurred in a STAC dose dependant manner, the sequences chosen naturally contain the necessary bulky hydrophobic at residues +1 and +6 positions. Substitution of these for alanine reduced or abolished activation.
In a second finding mutational analysis of SIRT1 identified an N terminal acidic residue E230 that when substituted did not affect SIRT1 activity but reduced STAC induced activation in this in vitro assay. This indicates that SIRT1 contains a STAC binding region around residue 230 that was critical for activation across 117 STACs of diverse structure. Therefore STACs act as allosteric activators of SIRT1. In mouse cell lines where SIRT1 was knocked out and replaced with a lysine substituted enzyme at this position the effect of STACs was blocked and an increase in mitochondrial mass, ATP, and mitochondrial DNA copy number was no longer observed when cells were treated with resveratrol or STAC-4.
Also this month, Lakshminarasimhan et al. independently confirmed the previous study's first finding that SIRT1 activation by STACs requires a proximal hydrophobic residue in the target sequence. Using a 6802 (fluorophore-free) acetylated peptide microarray, the acetylation level of each peptide was determined after SIRT1 and resveratrol incubation using an anti-acetyl-Lysine antibody. Resveratrol significantly increased the SIRT1 dependant deactylation of some peptides where large, mainly hydrophobic residues, are present at several positions C terminal to the acetylated lysine. Conversely hydrophilic N terminal, or polar and negatively charged residues C terminal are inhibitory to deactylation stimulation.
Evidence for a common mechanism of SIRT1 regulation by allosteric activators. Science 2013. Hubbard BP, Gomes AP, Dai H, Li J, Case AW, Considine T, Riera TV, Lee JE, E SY, Lamming DW, Pentelute BL, Schuman ER, Stevens LA, Ling AJ, Armour SM, Michan S, Zhao H, Jiang Y, Sweitzer SM, Blum CA, Disch JS, Ng PY, Howitz KT, Rolo AP, Hamuro Y, Moss J, Perni RB, Ellis JL, Vlasuk GP, Sinclair DA.
Sirt1 activation by resveratrol is substrate sequence-selective. Aging. 2013 Lakshminarasimhan M, Rauh D, Mike Schutkowski M, Steegborn C
Related papers using MitoSciences products this month:
Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression. J Clin Invest. 2013 Santidrian AF, Matsuno-Yagi A, Ritland M, Seo BB, Leboeuf SE, Gay LJ, Yagi T, Felding-Habermann B.
Mitochondrial SIRT4-type proteins in Caenorhabditis elegans and mammals interact with pyruvate carboxylase and other acetylated biotin-dependent carboxylases. Mitochondrion. 2013 Wirth M, Karaca S, Wenzel D, Ho L, Tishkoff D, Lombard DB, Verdin E, Urlaub H, Jedrusik-Bode M, Fischle W.
α-SNAP inhibits AMPK signaling to reduce mitochondrial biogenesis and dephosphorylates Thr172 in AMPKα in vitro. Nat Commun. 2013 Wang L, Brautigan DL.
AMPK is required for exercise-induced PGC-1α translocation to subsarcolemmal mitochondria in skeletal muscle. J Physiol. 2013 Smith BK, Mukai K, Lally JS, Maher AC, Gurd BJ, Heigenhauser GJ, Spriet LL, Holloway GP.
The Import of the Transcription Factor STAT3 into Mitochondria Depends on GRIM-19, a Component of the Electron Transport Chain. J Biol Chem. 2013 Tammineni P, Anugula C, Mohammed F, Anjaneyulu M, Larner AC, Sepuri NB.