Edited by James Murray, PhD.
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. The full list of 27 original research papers published this month using the MitoSciences range of products can be found here.
Past issues are available for review in the archives.
Thousands of acetylated mammalian proteins have been identified by proteomic methods. Studies using these techniques reveal that reversible acetyl modification, primarily of lysine residues, is not limited to histones, but is widely seen in, and regulates the activities of, proteins of metabolic pathways.
Sirtuins are a family of NAD+ dependant enzymes that act as deacetylases or ADP-ribosyl transferases to regulate these metabolic pathways. As result of their dependence on NAD+, their activity is directly linked to the energy status of the cell and the NAD+/NADH ratio. The effect of deacetylation or ADP-ribosyl transfer is primarily of protection against age related diseases including cancer, inflammation, cardiovascular disease, diabetes, neurodegeneration and in aging itself.
In humans and mice seven sirtuins exist (SIRT1-7) in multiple cellular locations. SIRT3, 4 and 5 have been identified as regulators of different mitochondrial functions. To date SIRT5 appears to have a specific role in ammonia detoxification during fasting by the deactylation dependant activation of mitochondrial CPS1. SIRT4 has no deactylase activity, but instead transfers ADP-ribosyl groups on to the mitochondrial glutamate dehydrogenase, GLUD1. This modification results in inactivation of the enzyme, downregulation of glutamate/ glutamine metabolism, and in turn reduces insulin secretion. While these two mitochondrial sirtuins have quite specific functions identified so far, the third mitochondrial sirtuin, SIRT3, appears to exert its effect more broadly by regulating a large number of mitochondrial pathways. SIRT3 is responsive to caloric restriction and promotes mitochondrial oxidative metabolism by deactylation dependant up regulation of oxidative phosphorylation, fatty acid oxidation pathways, and ketone body synthesis. SIRT3 also decreases reactive oxygen species by regulating the anti-oxidant enzyme mitochondrial Mn-SOD (SOD2) at the gene transcription level but also directly by deactylation dependant upregulation of the enzyme. Consequently SIRT3 activity has been implicated as protective in a number of diseases including neurodegeneration and cancer. Increased oxidative metabolism and removal of toxic ROS reduces neuronal sensitivity to apoptosis and toxic protein aggregation. In cancer these activities counteract the Hif1 alpha metabolic switch. This suggests that SIRT3 is a tumor suppressor and, in support of this, SIRT3 knockout mice have a higher degree of spontaneous mammary tumors.
This month,, Verma et al. strengthened the case for SIRT3 as a tumor suppressor. The authors propose that SIRT3 modulates apoptosis by regulating the Bak/Bax interaction with mitochondrial membranes in a cyclophilin D dependant manner. The authors showed that SIRT3 depletion makes cultured cells less sensitive to apoptosis and increases ROS which stabilizes Hif1 alpha and in turn induces hexokinase II expression. The authors propose that cyclophilin D activity is also increased by preventing SIRT3 dependant deactylation. Cyclophilin D is a key regulator of the permeability transition pore, the resulting increased activity of this enzyme promotes hexokinase II recruitment to the mitochondrial outer membrane and its interaction with VDAC. This interaction promotes apoptotic insensitivity by preventing an interaction between VDAC with Bak/Bax, a necessary step in the induction of mitochondrial outer membrane permeabilization (MOMP) in apoptosis.
Sirtuin-3 Modulates Bak/Bax Dependent Apoptosis. J Cell Sci 2012. Verma M, Shulga N, Pastorino JG.
Sirtuins have long been linked with aging and age related diseases. Mitochondrial function and the level of mtDNA in brain and heart decline with age. This month Thomas et al. published the result of their work attempting to increase mitochondrial function in aged rat brains by intravenous treatment with recombinant human mitochondrial transcription factor A (rhTFAM). Treatment improved brain function and mitochondrial respiration. At the molecular level, increases in expression of mitochondrial ribosomes and SIRT3 were observed. These changes likely led to the demonstrated increase in OXPHOS expression, Complex I and IV activity, and a decrease in oxidative stress induced lipid peroxidation products. The authors conclude that increased SIRT3 expression likely results in upregulation of respiratory chain activity and anti-oxidant enzyme activity by deacetylation of OXPHOS and SOD2, respectively.
RhTFAM treatment stimulates mitochondrial oxidative metabolism and improves memory in aged mice. Aging 2012. Thomas RR, Khan SM, Smigrodzki RM, Onyango IG, Dennis J, Khan OM, Portelli FR, Bennett JP Jr.
Finally, this month Wu et al. showed a novel regulation of the F1F0 ATP synthase in mouse mitochondria mediated by SIRT3. These researchers propose that acetylation of the OSCP stalk subunit destabilizes the F1F0, impairs energy coupling, and prevents supercomplex formation. They demonstrated a direct interaction between OSCP and SIRT3, which in turn promotes deactylation of neighboring subunits such as F1 alpha. In cells harboring mtDNA with the 4977 bp deletion, the researchers showed an oxidative stress dependant decrease in SIRT3 level, and an associated increase in acetylation of F1 alpha and OSCP.
Regulation of mitochondrial F(o)F(1)ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977bp deletion of mitochondrial DNA. BBA 2012. Davison R, Kaplan K, Fintel D, Parker M, Anderson L, Haring O.
Endurance training and chronic intermittent hypoxia modulate in vitro salicylate-induced hepatic mitochondrial dysfunction. Mitochondrion 2012. Ascensão A, Gonçalves IO, Lumini-Oliveira J, Marques-Aleixo I, Dos Passos E, Rocha-Rodrigues S, Machado NG, Moreira AC, Oliveira PJ, Torrella JR, Magalhães J.
Inhibition of Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances Oxidative Metabolism in Skeletal Muscle and Adipose Tissue. Diabetes 2012. Galmozzi A, Mitro N, Ferrari A, Gers E, Gilardi F, Godio C, Cermenati G, Gualerzi A, Donetti E, Rotili D, Valente S, Guerrini U, Caruso D, Mai A, Saez E, De Fabiani E, Crestani M.
p53 Induced Apoptosis WB Cocktail (ab140360)
Membrane Fractionation WB cocktail (ab140365)
AMPK alpha ELISA Kit (ab140358)
In Cell ELISA Kits
MitoBiogenesis (Fluorescent) (ab140359)
Cell cycle (Fluorescent) (ab140363)
Cleaved PARP1 (Fluorescent) (ab140362)
Lactate dehydrogenase B Activity Assay (ab140361)
Cyclophilin D (E11AE12) (ab110324)
VDAC1/Porin (20B12) (ab14734)
F1 alpha (ab14748)
Human OXPHOS WB Cocktail (ab110411)
Rodent OXPHOS WB Cocktail (ab110413)